一种NtHMA4基因突变体的制备方法与应用与流程

文档序号:11145327
一种NtHMA4基因突变体的制备方法与应用与制造工艺

本发明属于遗传工程技术领域,具体涉及一种NtHMA4突变体的制备方法与应用。



背景技术:

镉( Cd)、砷( As) 、铬( Cr) 、汞( Hg)和铅( Pb) 等是公认的对植物和人体危害比较大的重金属元素,植物必需微量元素锌( Zn) 、镍( Ni) 、铜( Cu)等如果过量也会产生危害。所以,关于作物重金属污染及其降低方法的研究越来越引起科研人员的重视。烟草(Nicotiana tabacum)是重要的经济作物,是茄科一年生草本植物。近年来关于烟草中重金属的研究也逐渐成为烟草减害研究的一个热点。

重金属对烟草品质会产生不利影响。烟碱含量是衡量烟叶品质的一个重要指标,在一定范围内提高烟碱有利于烟叶品质的提高,Cd 污染会降低烟叶烟碱含量,可能原因是根合成烟碱途径或者烟碱向上部转运过程受阻。另外,烟叶中可溶性糖对Cd、Pb极为敏感,轻度污染就会导致其含量明显下降,这可能是由于重金属破坏了烟草叶绿体,光合作用受阻引起的。重金属会导致烟叶糖碱比和氮碱比升高,化学成分组成失衡,品质受到影响。

重金属对人体的危害巨大,如过量的镉进入人体内可能对血管造成损害;另外镉元素对肠道吸收铁具有抑制作用,还会干扰钴、铜、锌等微量元素的代谢过程,引起肺、肾、肝等人体脏器损害,最终可能导致致癌、致畸和致突变等作用。砷可以诱发许多疾病,如急性砷中毒可造成中枢神经系统障碍导致全身麻木、呼吸道和消化道病变甚至快速死亡;慢性砷中毒导致神经系统紊乱、全身乏力、食欲减退、恶心以及皮肤色素沉着和角化病等皮肤病变等,而且可以引起肺癌、皮肤癌和膀胱癌等疾病。锌元素是人体的重要的微量元素之一,对人体的生长发育、智力、免疫功能甚至是视力都有至关重要的作用。但是人体中的锌不是越多越好。人体摄入过量的锌,在胃液中易转化成氯化锌,对胃黏膜有较强的腐蚀性,可致胃黏膜充血、水肿、甚至出血。过量的血锌会抑制白细胞的吞噬功能,是人体抵抗力下降,易受病菌感染。过量的锌还会影响人体其他无机盐的吸收与代谢,如影响铁的吸收,使肝脏中铁代谢受损成铜/锌比值过高;影响胆固醇代谢,形成高胆固醇血症等。镍及其盐类的毒性较低,但由于它本身具有生物化学活性,故能激活或抑制一系列的酶(精氨酸酶、羧化酶、酸性磷酸酶和拓脱羧酶)而发挥其毒性。镍可引起接触性皮炎。直接进入血液的镍盐毒性较高,胶体镍或氯化镍毒性较大,可引起中枢性循环和呼吸紊乱,使心肌、脑、肺和肾出现水肿、出血和变性。

烟草是我国重要的经济作物, 重金属污染不仅影响烟草本身生长发育,对吸食者的的健康也具有严重的威胁。减害一直是烟草科研工作的一个重要方向,其中降低重金属含量是一个重要的公关目标。



技术实现要素:

本发明的第一目的在于提供一种NtHMA4基因的EMS突变体;第二目的在于提供所述的NtHMA4基因突变体的应用。

本发明的第一目的是这样实现的,与野生型烟草的NtHMA4基因具有如序列表中SEQ ID No:1所示的核苷酸序列相比,突变体含有的NtHMA4基因序列152位的C突变为T,使该基因编码蛋白序列第51位氨基酸由苏氨酸(Thr)突变为异亮氨酸(Ile)。

所述的NtHMA4基因的EMS突变体的制备方法包括EMS诱变和TILLING筛选突变体。

EMS诱变烟草种子:

A :利用漂白水处理烟草种子,然后旋转离心并滤干;

B :对种子进行漂洗,清除漂白水,使种子不受漂白剂化学成分影响,然后进行旋转离心滤干;

C :将种子放在10℃ ~30℃温度下的去离子水中浸泡10~15小时,烟草种子催芽萌发,以利种子均匀诱变处理,然后进行旋转离心滤干;

D :将种子放在0.5%的EMS (甲基磺酸乙酯)诱变剂溶液中浸泡处理10~15个小时,然后进行旋转离心滤干;

E :将种子放在漂洗剂溶液中漂洗,再用去离子水冲洗,反复进行5~8 次此过程,然后布氏漏斗滤纸过滤干燥。

TILLING筛选核苷酸变化的突变体

A :诱变处理后的种子(M1代)播种与大田,单株套袋自交收种获得M2代,每个M1代单株收获的M2代种子播种1粒种子。

B:取M2代突变体单株叶片利用QIAGEN DNA提取试剂盒提取突变体材料叶片DNA。

C:样品浓度测定及建池

将样品按照顺序大小排列。分别取2ul DNA样品16通道 Tecan infinite M200仪器上进行浓度测定。将所有的样品浓度稀释至40ng/ul,制作8倍DNA池用于TILLING分析。

D: 利用Primer3设计NtHMA4基因TILLING分析引物,

HMA4-F:TAGAGTGTAGAGGAAAAATAGAAAGAAGAG和

HMA4-R: ATAAGCTGAGAGCTTAAGAAAAAAGAAACT,扩增长度为500bp左右。

E:TILLING分析 M2代突变体,筛选核苷酸突变的单株,并进行测序验证,获得一个突变体,其CDS的152位的C突变为T,使该基因编码蛋白序列第51位氨基酸由苏氨酸(Thr)突变为异亮氨酸(Ile)。

F:在M3代突变体筛选突变位点纯合的植株,自交收种。

G:对纯合突变体进行镉吸收试验,具体为在烟株现蕾期,将100µmol CdCL2溶液500mL浇于烟株根部。5天后取叶片,杀青烘干,检测重金属含量。

H: 含有突变序列的烟草相比含有SEQ ID NO:1序列的烟草叶片镉含量降低50%左右;铜、锌、镍降低40%左右;铁、锰降低30%左右;铅、铬降低20%左右。

本发明的第二目的是这样实现的,NtHMA4基因突变体可以使烟叶镉含量降低40~60%;铜、锌、镍降低30~50%;铁、锰降低20~40%;铅、铬降低10~30%。即以所述的NtHMA2基因突变体获得低重金属含量的烟草。

附图说明

图1 为突变体突变位点测序峰图;

图2为本发明NtHMA4突变体与对照叶片镉含量对比示意图;

图3为本发明NtHMA4突变体与对照叶片铜含量对比示意图;

图4为本发明NtHMA4突变体与对照叶片锌含量对比示意图;

图5为本发明NtHMA4突变体与对照叶片铁含量对比示意图;

图6为本发明NtHMA4突变体与对照叶片锰含量对比示意图;

图7为本发明NtHMA4突变体与对照叶片铅含量对比示意图;

图8为本发明NtHMA4突变体与对照叶片铬含量对比示意图;

图9为本发明NtHMA4突变体与对照叶片镍含量对比示意图。

具体实施方式

下面结合实施例和附图对本发明作进一步的说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变换或替换,均属于本发明的保护范围。

与野生型烟草的NtHMA4基因具有如序列表中SEQ ID No:1所示的核苷酸序列相比,突变体含有的NtHMA4基因序列152位的C突变为T,使该基因编码蛋白序列第51位氨基酸由苏氨酸(Thr)突变为异亮氨酸(Ile)。

NtHMA4基因编码的氨基酸序列如SEQ ID No:2所示;

NtHMA4基因突变体的核苷酸序列如SEQ ID No:3所示;

NtHMA4基因突变体编码的氨基酸序列如SEQ ID No:4所示;

所述的NtHMA4基因的EMS突变体的制备方法包括EMS诱变和TILLING筛选突变体。

EMS诱变烟草种子:

A :利用漂白水处理烟草种子,然后旋转离心并滤干;

B :对种子进行漂洗,清除漂白水,使种子不受漂白剂化学成分影响,然后进行旋转离心滤干;

C :将种子放在10℃ ~30℃温度下的去离子水中浸泡10~15小时,烟草种子催芽萌发,以利种子均匀诱变处理,然后进行旋转离心滤干;

D :将种子放在0.5%的EMS (甲基磺酸乙酯)诱变剂溶液中浸泡处理10~15个小时,然后进行旋转离心滤干;

E :将种子放在漂洗剂溶液中漂洗,再用去离子水冲洗,反复进行5~8 次此过程,然后布氏漏斗滤纸过滤干燥。

TILLING筛选核苷酸变化的突变体

A :诱变处理后的种子(M1代)播种与大田,单株套袋自交收种获得M2代,每个M1代单株收获的M2代种子播种1粒种子。

B:取M2代突变体单株叶片利用QIAGEN DNA提取试剂盒提取突变体材料叶片DNA。

C:样品浓度测定及建池

将样品按照顺序大小排列。分别取2ul DNA样品16通道 Tecan infinite M200仪器上进行浓度测定。将所有的样品浓度稀释至40ng/ul,制作8倍DNA池用于TILLING分析。

D: 利用Primer3设计NtHMA4基因TILLING分析引物,

HMA4-F:TAGAGTGTAGAGGAAAAATAGAAAGAAGAG和

HMA4-R: ATAAGCTGAGAGCTTAAGAAAAAAGAAACT,扩增长度为500bp左右。

E:TILLING分析 M2代突变体,筛选核苷酸突变的单株,并进行测序验证,获得一个突变体,其CDS的152位的C突变为T,使该基因编码蛋白序列第51位氨基酸由苏氨酸(Thr)突变为异亮氨酸(Ile)。

F:在M3代突变体筛选突变位点纯合的植株,自交收种。

G:对纯合突变体进行镉吸收试验,具体为在烟株现蕾期,将100µmol CdCL2溶液500mL浇于烟株根部。5天后取叶片,杀青烘干,检测重金属含量。

H: 含有突变序列的烟草相比含有SEQ ID NO:1序列的烟草叶片镉含量降低50%左右;铜、锌、镍降低40%左右;铁、锰降低30%左右;铅、铬降低20%左右。

本发明的第二目的是这样实现的,NtHMA4基因突变体可以使烟叶镉含量降低40~60%;铜、锌、镍降低30~50%;铁、锰降低20~40%;铅、铬降低10~30%。即以所述的NtHMA2基因突变体获得低重金属含量的烟草。

下面以具体实施案例对本发明做作进一步说明:

所述的NtHMA4突变体的制备方法, 未突变NtHMA4突变体的核苷酸序列如序列表中SEQ ID No:1所示,突变体的核苷酸序列152位的C突变为T,具体制备方法包括以下步骤:

(1)选用50% 漂白水处理云烟87种子6 分钟,离心滤干;

(2)利用去离子对种子进行漂洗1 分钟,离心滤干去除漂白水成分;

(3)室温条件,去离子水浸泡烟草种子10-12 小时,离心滤干;

(4)室温条件,0.50%EMS 甲基磺酸乙酯处理烟草种子12 小时,离心滤干;

(5)加入去离子水漂洗1 分钟,离心滤干,共重复漂洗8次。然后布氏漏斗滤纸过滤干燥。

(6)诱变处理后的种子(M1代)播种与大田,单株套袋自交收种获得M2代,每个M1代单株收获的M2代种子播种1粒种子,共获得1842个M2代单株。

(7) 取M2代突变体单株叶片利用QIAGEN DNA提取试剂盒提取突变体材料叶片DNA。具体步骤如下:将样品液氮研磨,并用1.5ml离心管收取;加入400ul的AP1溶液,4ul的RNA酶混匀 ;把样品放入65℃水浴锅水浴10min,期间摇匀3次;往样品中加入130ul P3溶液,冰浴5mim;高速离心,13200rpm,5min;取上层液倒入紫色过滤柱,13200rpm,2min离心;将滤液转入新的1.5mL离心管并加入675ul的AW1溶液混匀;把混匀样品溶液分2次转入淡黄色过滤柱并进行10000rpm,1min离心,弃除滤液;把淡黄色过滤柱放入新的2ml离心管中并加入500ul的AW2溶液,进行10000rpm,1min离心,弃滤液 (此步骤重复两次);再把弃除滤液的淡黄色过滤柱进行空转(10000rpm,1min),将空转好的淡黄色过滤柱放入新的1.5ml离心管中加入50ul AE溶液后静置5min,放入离心机进行10000rpm,1min离心,去除淡黄色过滤柱,放入4℃冰箱保存。

(8)样品浓度测定及建池

将样品按照顺序大小排列。分别取2ul DNA样品16通道 Tecan infinite M200仪器上进行浓度测定。将所有的样品浓度稀释至40ng/ul,制作8倍DNA池用于TILLING分析。

(9)利用Primer3设计NtHMA4基因TILLING分析引物,

HMA4-F:TAGAGTGTAGAGGAAAAATAGAAAGAAGAG和

HMA4-R: ATAAGCTGAGAGCTTAAGAAAAAAGAAACT,扩增长对为500bp左右。

(10)TILLING分析 M2代突变体,扩增体系为:1.0µl 10×buffer,0.8µl dNTP(2.5mM),0.16 µl HMA4-F primer(10µM) ,0.16µl HMA4R primer (10uM), 6.78 µl H2O,1.0 µl DNA模板 (20ng/ul) 。反应程序为95℃ 3min,94℃ 30s,62℃ 30s,-1℃/循环,72℃ 1min,7个循环;94℃30s,58℃30s,72℃1min,40个循环,72℃5min;99℃ 10min;70℃20s, -0.3℃/循环, 70个循环,4℃保存。扩增产物通过毛细管电泳进行分析。

(10)筛选出的核苷酸突变的单株,进行测序验证,获得一个突变体,其NtHMA4基因CDS的152位的C突变为T,使该基因编码蛋白序列第51位氨基酸由苏氨酸(Thr)突变为异亮氨酸(Ile)。

(11)在M3代突变体中经过测序筛选突变位点纯合的植株(图1),自交收种。

本发明所述的制备的NtHMA4突变体的应用为NtHMA4突变体用于降低烟叶重金属含量。

NtHMA4突变体降低烟叶重金属含量的方法包括以下步骤:

(1)对纯合突变体进行镉吸收试验,具体为在温室种植纯合突变体及野生型对照,烟株现蕾期,将100µmol CdCL2溶液500mL浇于烟株根部,5天后取叶片,杀青烘干。利用烟草行业标准YC/T380-2010的方法检测烟叶重金属含量。

(2)含有突变序列的烟草相比含有SEQ ID NO:1序列的烟草叶片镉含量降低50%左右;铜、锌、镍降低40%左右;铁、锰降低30%左右;铅、铬降低20%左右。(表1,图2~图9)。

表1 烟叶重金属含量

SEQUENCE LISTING

<110> 云南省烟草农业科学研究院

<120> 一种NtHMA4基因突变体与应用

<130> 2017

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 4335

<212> DNA

<213> NtHMA4基因核苷酸

<400> 1

atggtggaaa gtgagaaaat gaatgacaca aagaatctga gcaagagcta ttttgatgtt 60

ttgggaattt gctgtacttc agaagttgtt cttgttgaaa aaattctcaa gaatcttgaa 120

ggggttaaag aggtttcagt aattgtcaca acaaagactg tcattgttat tcatgattct 180

ctcctcattt ctcagcaaca aattgttaaa gcattgaatc aagcaagatt agaagcaagt 240

ataagagtga aaggagagaa aaactaccaa aagaaatggc caagtccatt tgcaattggc 300

agtggaatat tgcttggact ctcatttttg aagtactttt ttgcaccttt ccaatggtta 360

gcacttgcag ctgttgcagt tgggattcct ccaattattt ttaggggtgt ggctgccgtg 420

cgaaacctca ctcttgacat caacattctt gttttaatag cagtgacggg atcaattgtt 480

ttacacgatt attgggaagc tggtactatt gtcttcttat tcaccattgc agaatggcta 540

gagtcaaggg caagtcacaa ggctactgct gctatgtcat cactggtcaa tatagtccct 600

ccaacagcag ttttagctga aagtggagaa gtcgtaaatg ttgatgaagt caagttgaat 660

agcattcttg ctgttaaagc tggtgaaact atacctattg atggagttgt aatggaaggg 720

gaatgtgacg tggacgagaa aacactgaca ggcgagtcgt ttccagtttc taagcaaata 780

gattcaacgg tctgggctgg cactacaaat ctaaatggct atatcagtgt taagactacg 840

gctttggctg aagattgtgc ggtggctagg atggcgcagc ttgtcgaaga tgctcagaac 900

aagaaatcaa aaacccaaag atacattgac aagtgtgcta aatattatac accagcaatt 960

gtggctatat cagcttcttt ggcaatagtt cctactgcat taagagttca caatcgaaat 1020

gagtggtatc gcttggcttt ggtcacgttg gtgagtgcat gtccgtgtgc acttgtgcta 1080

tctacaccag ttgccatgtg ttgtgcactt tctaaagcag caacgtccgg tcttctgttt 1140

aaaggagcag agtaccttga gactcttgct aaaatcaaaa tcatggcttt tgacaaaaca 1200

gggactataa ctagaggaga atttatggtg accgagttca agtctctggt tgatggtctt 1260

ggtctcaata cactgcttta ctgggtttca agtattgaga gcaagtcagg tcatccgatg 1320

gcagccgctc tggttgacta tgcacaatca aattccgttg agccaaagcc tgatagagtt 1380

gagcagtttc aaaattttcc tggtgaaggg atatttggaa gaattgatgg aatggaaatc 1440

tatgtcggga ataggaaaat ttcttcaaga gctggatgta ctacagtacc agaaatagag 1500

ggtgatagtt tccaaggaaa gtctgttgga tacatatttt tgggatcatc tcccgctgga 1560

attttcggtc tttccgatgt ttgtcgaatt ggtgtaaaag aagccatgag agagctgaag 1620

cagatgggta tcaaaaccgc gatgcttact ggtgattgtt atgcagctgc caaccatgtg 1680

caggatcagt taggtggagc tatggatgaa tttcaagcgg aactcttacc agaggacaag 1740

gcaacaatca tcaagggttt tcagaaggaa gctccaacag cgatgatagg cgacggcctt 1800

aatgatgctc ctgcattagc aacagctgac attggcatct caatgggcat ctctgggtca 1860

gctctcgcga aagaaacagg ccatgttata ctaatgacaa atgacatcgg aagaatacca 1920

aaagctgcac gtcttgctag aagagttcga aggaagattg ttgagaatat gattatatca 1980

gtcgttacaa aggccgccat agttgcattg gcaatagcag gttatccatt ggtttgggct 2040

gctgtcctcg cggatactgg gacatgcttg ctagtgatct tgaacagcat gctacttcta 2100

cgagtaggca cacacagaca tgggaaaaaa tgttgtagat ctgctactcc ttcgcatgct 2160

cccaaccaca aagacaaagc ttcttgttgc aagtcggaaa atgctccgca gctgtgttgc 2220

tctgatattg agtcacaaaa gaaatgtacg agtcaatcat gctcgtccga ggtgtgtgtt 2280

ccaagatgtc aacctgtctc ctcgggatca aagtcatgtg gaaataatca gtgcccagac 2340

tccgttgaaa atagtggttt tcattctcat ccccgtcctc aatgctgctc gtcgaagatg 2400

gcttctaaag catgccaatc tgcagtttca gaatcaaagt catgtggaaa taatcagtgc 2460

ccagactccg ttgaaaatag tggttttcat tctcatcccc gccctcaatg ctgctcgtct 2520

aagatggctt ctaaagcatg ccaatctgca gtttcagaat caaagtcatg tggaaataat 2580

cagtgcccag actccgttga aaatagtggt tttcattctc atccccgtcc tcaatgctgc 2640

tcgttgaaga tggcttctaa agcatgccaa tctgcagttt cagaatcaaa gtcatgtgga 2700

aataatcagt gcccagactc cgttgaaaat agtggttttc attctcatcc ccgtcctcaa 2760

tgctgctcgt cgaagatggc tgctaaagca tgccaatctg cagtttcaga atcaaagtca 2820

tgtggaaata acaattgctc ggagtccatt tacaagagta gttgtcattc tttaacaagt 2880

tctctagtat gttcttccaa gatgtctgct ccacaatgtc attctgccac ttcaagctcc 2940

aaatcatgtg gaagtaccaa gtgctccaac ttcagtgaca aaaaatgttg ccaatatgac 3000

aaaattcctc aaacgtgctc taccaagaag tctgctccag gatgtcaatc tgcagtttct 3060

gggtctaaat catgtggaga tagcaagtgt tcagactcga aagacaatag tagccatcct 3120

tcacatcccg atcatcaaat atgcacgtct aagttgtgtg ctccacaaag ccaatctgca 3180

acttcaagct ccaggacatg tggaaatatg aagtgctcgg acaccaatag caagaattct 3240

tgttattcac ataccaactc tgaatcatgc tcttcaaaga tgtctggtcc agcatgcaaa 3300

actgctaatt caggttcaag gttatgcgga aataagaagt gcctagactc tgcaaacgag 3360

aacagttttc attcacttac taatccactc tgtgaggaaa agcttttgga gaaggaaagc 3420

ttggatttag cccgaaaaga tagggaatca aatcatgatc ttagtcatgg ttgctctgac 3480

gaggaacatg atcatctaaa tttagacaag gcacatgaca gttgtgcctt acaagaatgt 3540

tgttattctg ttcaaggcaa taaaactgat gtatcagaaa ctggaatcca ggaagctgct 3600

cattgtgaca gcatcaatca aacatgccaa actgcaattt caggatcaat gacatgcgga 3660

aataataaga gtctggactc tctaagcatc catggttgtc attcacatga tagtccactc 3720

cacaaggaga gcaacttgga gcagaaaagc ttggatgttg ctggagaagg tataaaatca 3780

cctcatgctg tcggtcaagg ctgttcggac aaggagcaca atcactcgca tccagaaaag 3840

gcgtatgaca gttgtgcaac agacgattgt tgtttttcag ttcaagtcca tggcattgac 3900

gacgtatcaa gaagtgaaat tcaagaaact gctcattgtg acagcacaaa acagagcacg 3960

gtcatcccca gcagctgcga acatgaacca aaagatcagg taaatcactg tggatctcac 4020

tctaaaagta ttccaactga tgaagaacta gccaagctgg ttagaagatg ctgcaaatac 4080

aaaccatgcc acgatgtccg ctctggctgc aggaagcatg ctgcagaatg tggtccaacc 4140

gttcgatcaa ccatcaatat cttacgggac aaccatcatc atcatctaga ctgcagtggt 4200

cgtaaggttt gttcgctgtt ggagaagaga cacattggtg gatgctgtga cagcttcaga 4260

aaagaatgtt gtgccaagaa caatcacctt ggagcaagtt ttggaggagg tttatcagaa 4320

attgtcatag agtag 4335

<210> 2

<211> 1444

<212> PRT

<213> NtHMA4基因氨基酸

<400> 2

Met Val Glu Ser Glu Lys Met Asn Asp Thr Lys Asn Leu Ser Lys Ser

1 5 10 15

Tyr Phe Asp Val Leu Gly Ile Cys Cys Thr Ser Glu Val Val Leu Val

20 25 30

Glu Lys Ile Leu Lys Asn Leu Glu Gly Val Lys Glu Val Ser Val Ile

35 40 45

Val Thr Thr Lys Thr Val Ile Val Ile His Asp Ser Leu Leu Ile Ser

50 55 60

Gln Gln Gln Ile Val Lys Ala Leu Asn Gln Ala Arg Leu Glu Ala Ser

65 70 75 80

Ile Arg Val Lys Gly Glu Lys Asn Tyr Gln Lys Lys Trp Pro Ser Pro

85 90 95

Phe Ala Ile Gly Ser Gly Ile Leu Leu Gly Leu Ser Phe Leu Lys Tyr

100 105 110

Phe Phe Ala Pro Phe Gln Trp Leu Ala Leu Ala Ala Val Ala Val Gly

115 120 125

Ile Pro Pro Ile Ile Phe Arg Gly Val Ala Ala Val Arg Asn Leu Thr

130 135 140

Leu Asp Ile Asn Ile Leu Val Leu Ile Ala Val Thr Gly Ser Ile Val

145 150 155 160

Leu His Asp Tyr Trp Glu Ala Gly Thr Ile Val Phe Leu Phe Thr Ile

165 170 175

Ala Glu Trp Leu Glu Ser Arg Ala Ser His Lys Ala Thr Ala Ala Met

180 185 190

Ser Ser Leu Val Asn Ile Val Pro Pro Thr Ala Val Leu Ala Glu Ser

195 200 205

Gly Glu Val Val Asn Val Asp Glu Val Lys Leu Asn Ser Ile Leu Ala

210 215 220

Val Lys Ala Gly Glu Thr Ile Pro Ile Asp Gly Val Val Met Glu Gly

225 230 235 240

Glu Cys Asp Val Asp Glu Lys Thr Leu Thr Gly Glu Ser Phe Pro Val

245 250 255

Ser Lys Gln Ile Asp Ser Thr Val Trp Ala Gly Thr Thr Asn Leu Asn

260 265 270

Gly Tyr Ile Ser Val Lys Thr Thr Ala Leu Ala Glu Asp Cys Ala Val

275 280 285

Ala Arg Met Ala Gln Leu Val Glu Asp Ala Gln Asn Lys Lys Ser Lys

290 295 300

Thr Gln Arg Tyr Ile Asp Lys Cys Ala Lys Tyr Tyr Thr Pro Ala Ile

305 310 315 320

Val Ala Ile Ser Ala Ser Leu Ala Ile Val Pro Thr Ala Leu Arg Val

325 330 335

His Asn Arg Asn Glu Trp Tyr Arg Leu Ala Leu Val Thr Leu Val Ser

340 345 350

Ala Cys Pro Cys Ala Leu Val Leu Ser Thr Pro Val Ala Met Cys Cys

355 360 365

Ala Leu Ser Lys Ala Ala Thr Ser Gly Leu Leu Phe Lys Gly Ala Glu

370 375 380

Tyr Leu Glu Thr Leu Ala Lys Ile Lys Ile Met Ala Phe Asp Lys Thr

385 390 395 400

Gly Thr Ile Thr Arg Gly Glu Phe Met Val Thr Glu Phe Lys Ser Leu

405 410 415

Val Asp Gly Leu Gly Leu Asn Thr Leu Leu Tyr Trp Val Ser Ser Ile

420 425 430

Glu Ser Lys Ser Gly His Pro Met Ala Ala Ala Leu Val Asp Tyr Ala

435 440 445

Gln Ser Asn Ser Val Glu Pro Lys Pro Asp Arg Val Glu Gln Phe Gln

450 455 460

Asn Phe Pro Gly Glu Gly Ile Phe Gly Arg Ile Asp Gly Met Glu Ile

465 470 475 480

Tyr Val Gly Asn Arg Lys Ile Ser Ser Arg Ala Gly Cys Thr Thr Val

485 490 495

Pro Glu Ile Glu Gly Asp Ser Phe Gln Gly Lys Ser Val Gly Tyr Ile

500 505 510

Phe Leu Gly Ser Ser Pro Ala Gly Ile Phe Gly Leu Ser Asp Val Cys

515 520 525

Arg Ile Gly Val Lys Glu Ala Met Arg Glu Leu Lys Gln Met Gly Ile

530 535 540

Lys Thr Ala Met Leu Thr Gly Asp Cys Tyr Ala Ala Ala Asn His Val

545 550 555 560

Gln Asp Gln Leu Gly Gly Ala Met Asp Glu Phe Gln Ala Glu Leu Leu

565 570 575

Pro Glu Asp Lys Ala Thr Ile Ile Lys Gly Phe Gln Lys Glu Ala Pro

580 585 590

Thr Ala Met Ile Gly Asp Gly Leu Asn Asp Ala Pro Ala Leu Ala Thr

595 600 605

Ala Asp Ile Gly Ile Ser Met Gly Ile Ser Gly Ser Ala Leu Ala Lys

610 615 620

Glu Thr Gly His Val Ile Leu Met Thr Asn Asp Ile Gly Arg Ile Pro

625 630 635 640

Lys Ala Ala Arg Leu Ala Arg Arg Val Arg Arg Lys Ile Val Glu Asn

645 650 655

Met Ile Ile Ser Val Val Thr Lys Ala Ala Ile Val Ala Leu Ala Ile

660 665 670

Ala Gly Tyr Pro Leu Val Trp Ala Ala Val Leu Ala Asp Thr Gly Thr

675 680 685

Cys Leu Leu Val Ile Leu Asn Ser Met Leu Leu Leu Arg Val Gly Thr

690 695 700

His Arg His Gly Lys Lys Cys Cys Arg Ser Ala Thr Pro Ser His Ala

705 710 715 720

Pro Asn His Lys Asp Lys Ala Ser Cys Cys Lys Ser Glu Asn Ala Pro

725 730 735

Gln Leu Cys Cys Ser Asp Ile Glu Ser Gln Lys Lys Cys Thr Ser Gln

740 745 750

Ser Cys Ser Ser Glu Val Cys Val Pro Arg Cys Gln Pro Val Ser Ser

755 760 765

Gly Ser Lys Ser Cys Gly Asn Asn Gln Cys Pro Asp Ser Val Glu Asn

770 775 780

Ser Gly Phe His Ser His Pro Arg Pro Gln Cys Cys Ser Ser Lys Met

785 790 795 800

Ala Ser Lys Ala Cys Gln Ser Ala Val Ser Glu Ser Lys Ser Cys Gly

805 810 815

Asn Asn Gln Cys Pro Asp Ser Val Glu Asn Ser Gly Phe His Ser His

820 825 830

Pro Arg Pro Gln Cys Cys Ser Ser Lys Met Ala Ser Lys Ala Cys Gln

835 840 845

Ser Ala Val Ser Glu Ser Lys Ser Cys Gly Asn Asn Gln Cys Pro Asp

850 855 860

Ser Val Glu Asn Ser Gly Phe His Ser His Pro Arg Pro Gln Cys Cys

865 870 875 880

Ser Leu Lys Met Ala Ser Lys Ala Cys Gln Ser Ala Val Ser Glu Ser

885 890 895

Lys Ser Cys Gly Asn Asn Gln Cys Pro Asp Ser Val Glu Asn Ser Gly

900 905 910

Phe His Ser His Pro Arg Pro Gln Cys Cys Ser Ser Lys Met Ala Ala

915 920 925

Lys Ala Cys Gln Ser Ala Val Ser Glu Ser Lys Ser Cys Gly Asn Asn

930 935 940

Asn Cys Ser Glu Ser Ile Tyr Lys Ser Ser Cys His Ser Leu Thr Ser

945 950 955 960

Ser Leu Val Cys Ser Ser Lys Met Ser Ala Pro Gln Cys His Ser Ala

965 970 975

Thr Ser Ser Ser Lys Ser Cys Gly Ser Thr Lys Cys Ser Asn Phe Ser

980 985 990

Asp Lys Lys Cys Cys Gln Tyr Asp Lys Ile Pro Gln Thr Cys Ser Thr

995 1000 1005

Lys Lys Ser Ala Pro Gly Cys Gln Ser Ala Val Ser Gly Ser Lys

1010 1015 1020

Ser Cys Gly Asp Ser Lys Cys Ser Asp Ser Lys Asp Asn Ser Ser

1025 1030 1035

His Pro Ser His Pro Asp His Gln Ile Cys Thr Ser Lys Leu Cys

1040 1045 1050

Ala Pro Gln Ser Gln Ser Ala Thr Ser Ser Ser Arg Thr Cys Gly

1055 1060 1065

Asn Met Lys Cys Ser Asp Thr Asn Ser Lys Asn Ser Cys Tyr Ser

1070 1075 1080

His Thr Asn Ser Glu Ser Cys Ser Ser Lys Met Ser Gly Pro Ala

1085 1090 1095

Cys Lys Thr Ala Asn Ser Gly Ser Arg Leu Cys Gly Asn Lys Lys

1100 1105 1110

Cys Leu Asp Ser Ala Asn Glu Asn Ser Phe His Ser Leu Thr Asn

1115 1120 1125

Pro Leu Cys Glu Glu Lys Leu Leu Glu Lys Glu Ser Leu Asp Leu

1130 1135 1140

Ala Arg Lys Asp Arg Glu Ser Asn His Asp Leu Ser His Gly Cys

1145 1150 1155

Ser Asp Glu Glu His Asp His Leu Asn Leu Asp Lys Ala His Asp

1160 1165 1170

Ser Cys Ala Leu Gln Glu Cys Cys Tyr Ser Val Gln Gly Asn Lys

1175 1180 1185

Thr Asp Val Ser Glu Thr Gly Ile Gln Glu Ala Ala His Cys Asp

1190 1195 1200

Ser Ile Asn Gln Thr Cys Gln Thr Ala Ile Ser Gly Ser Met Thr

1205 1210 1215

Cys Gly Asn Asn Lys Ser Leu Asp Ser Leu Ser Ile His Gly Cys

1220 1225 1230

His Ser His Asp Ser Pro Leu His Lys Glu Ser Asn Leu Glu Gln

1235 1240 1245

Lys Ser Leu Asp Val Ala Gly Glu Gly Ile Lys Ser Pro His Ala

1250 1255 1260

Val Gly Gln Gly Cys Ser Asp Lys Glu His Asn His Ser His Pro

1265 1270 1275

Glu Lys Ala Tyr Asp Ser Cys Ala Thr Asp Asp Cys Cys Phe Ser

1280 1285 1290

Val Gln Val His Gly Ile Asp Asp Val Ser Arg Ser Glu Ile Gln

1295 1300 1305

Glu Thr Ala His Cys Asp Ser Thr Lys Gln Ser Thr Val Ile Pro

1310 1315 1320

Ser Ser Cys Glu His Glu Pro Lys Asp Gln Val Asn His Cys Gly

1325 1330 1335

Ser His Ser Lys Ser Ile Pro Thr Asp Glu Glu Leu Ala Lys Leu

1340 1345 1350

Val Arg Arg Cys Cys Lys Tyr Lys Pro Cys His Asp Val Arg Ser

1355 1360 1365

Gly Cys Arg Lys His Ala Ala Glu Cys Gly Pro Thr Val Arg Ser

1370 1375 1380

Thr Ile Asn Ile Leu Arg Asp Asn His His His His Leu Asp Cys

1385 1390 1395

Ser Gly Arg Lys Val Cys Ser Leu Leu Glu Lys Arg His Ile Gly

1400 1405 1410

Gly Cys Cys Asp Ser Phe Arg Lys Glu Cys Cys Ala Lys Asn Asn

1415 1420 1425

His Leu Gly Ala Ser Phe Gly Gly Gly Leu Ser Glu Ile Val Ile

1430 1435 1440

Glu

<210> 3

<211> 4335

<212> DNA

<213> NtHMA4基因突变体核苷酸

<400> 3

atggtggaaa gtgagaaaat gaatgacaca aagaatctga gcaagagcta ttttgatgtt 60

ttgggaattt gctgtacttc agaagttgtt cttgttgaaa aaattctcaa gaatcttgaa 120

ggggttaaag aggtttcagt aattgtcaca ataaagactg tcattgttat tcatgattct 180

ctcctcattt ctcagcaaca aattgttaaa gcattgaatc aagcaagatt agaagcaagt 240

ataagagtga aaggagagaa aaactaccaa aagaaatggc caagtccatt tgcaattggc 300

agtggaatat tgcttggact ctcatttttg aagtactttt ttgcaccttt ccaatggtta 360

gcacttgcag ctgttgcagt tgggattcct ccaattattt ttaggggtgt ggctgccgtg 420

cgaaacctca ctcttgacat caacattctt gttttaatag cagtgacggg atcaattgtt 480

ttacacgatt attgggaagc tggtactatt gtcttcttat tcaccattgc agaatggcta 540

gagtcaaggg caagtcacaa ggctactgct gctatgtcat cactggtcaa tatagtccct 600

ccaacagcag ttttagctga aagtggagaa gtcgtaaatg ttgatgaagt caagttgaat 660

agcattcttg ctgttaaagc tggtgaaact atacctattg atggagttgt aatggaaggg 720

gaatgtgacg tggacgagaa aacactgaca ggcgagtcgt ttccagtttc taagcaaata 780

gattcaacgg tctgggctgg cactacaaat ctaaatggct atatcagtgt taagactacg 840

gctttggctg aagattgtgc ggtggctagg atggcgcagc ttgtcgaaga tgctcagaac 900

aagaaatcaa aaacccaaag atacattgac aagtgtgcta aatattatac accagcaatt 960

gtggctatat cagcttcttt ggcaatagtt cctactgcat taagagttca caatcgaaat 1020

gagtggtatc gcttggcttt ggtcacgttg gtgagtgcat gtccgtgtgc acttgtgcta 1080

tctacaccag ttgccatgtg ttgtgcactt tctaaagcag caacgtccgg tcttctgttt 1140

aaaggagcag agtaccttga gactcttgct aaaatcaaaa tcatggcttt tgacaaaaca 1200

gggactataa ctagaggaga atttatggtg accgagttca agtctctggt tgatggtctt 1260

ggtctcaata cactgcttta ctgggtttca agtattgaga gcaagtcagg tcatccgatg 1320

gcagccgctc tggttgacta tgcacaatca aattccgttg agccaaagcc tgatagagtt 1380

gagcagtttc aaaattttcc tggtgaaggg atatttggaa gaattgatgg aatggaaatc 1440

tatgtcggga ataggaaaat ttcttcaaga gctggatgta ctacagtacc agaaatagag 1500

ggtgatagtt tccaaggaaa gtctgttgga tacatatttt tgggatcatc tcccgctgga 1560

attttcggtc tttccgatgt ttgtcgaatt ggtgtaaaag aagccatgag agagctgaag 1620

cagatgggta tcaaaaccgc gatgcttact ggtgattgtt atgcagctgc caaccatgtg 1680

caggatcagt taggtggagc tatggatgaa tttcaagcgg aactcttacc agaggacaag 1740

gcaacaatca tcaagggttt tcagaaggaa gctccaacag cgatgatagg cgacggcctt 1800

aatgatgctc ctgcattagc aacagctgac attggcatct caatgggcat ctctgggtca 1860

gctctcgcga aagaaacagg ccatgttata ctaatgacaa atgacatcgg aagaatacca 1920

aaagctgcac gtcttgctag aagagttcga aggaagattg ttgagaatat gattatatca 1980

gtcgttacaa aggccgccat agttgcattg gcaatagcag gttatccatt ggtttgggct 2040

gctgtcctcg cggatactgg gacatgcttg ctagtgatct tgaacagcat gctacttcta 2100

cgagtaggca cacacagaca tgggaaaaaa tgttgtagat ctgctactcc ttcgcatgct 2160

cccaaccaca aagacaaagc ttcttgttgc aagtcggaaa atgctccgca gctgtgttgc 2220

tctgatattg agtcacaaaa gaaatgtacg agtcaatcat gctcgtccga ggtgtgtgtt 2280

ccaagatgtc aacctgtctc ctcgggatca aagtcatgtg gaaataatca gtgcccagac 2340

tccgttgaaa atagtggttt tcattctcat ccccgtcctc aatgctgctc gtcgaagatg 2400

gcttctaaag catgccaatc tgcagtttca gaatcaaagt catgtggaaa taatcagtgc 2460

ccagactccg ttgaaaatag tggttttcat tctcatcccc gccctcaatg ctgctcgtct 2520

aagatggctt ctaaagcatg ccaatctgca gtttcagaat caaagtcatg tggaaataat 2580

cagtgcccag actccgttga aaatagtggt tttcattctc atccccgtcc tcaatgctgc 2640

tcgttgaaga tggcttctaa agcatgccaa tctgcagttt cagaatcaaa gtcatgtgga 2700

aataatcagt gcccagactc cgttgaaaat agtggttttc attctcatcc ccgtcctcaa 2760

tgctgctcgt cgaagatggc tgctaaagca tgccaatctg cagtttcaga atcaaagtca 2820

tgtggaaata acaattgctc ggagtccatt tacaagagta gttgtcattc tttaacaagt 2880

tctctagtat gttcttccaa gatgtctgct ccacaatgtc attctgccac ttcaagctcc 2940

aaatcatgtg gaagtaccaa gtgctccaac ttcagtgaca aaaaatgttg ccaatatgac 3000

aaaattcctc aaacgtgctc taccaagaag tctgctccag gatgtcaatc tgcagtttct 3060

gggtctaaat catgtggaga tagcaagtgt tcagactcga aagacaatag tagccatcct 3120

tcacatcccg atcatcaaat atgcacgtct aagttgtgtg ctccacaaag ccaatctgca 3180

acttcaagct ccaggacatg tggaaatatg aagtgctcgg acaccaatag caagaattct 3240

tgttattcac ataccaactc tgaatcatgc tcttcaaaga tgtctggtcc agcatgcaaa 3300

actgctaatt caggttcaag gttatgcgga aataagaagt gcctagactc tgcaaacgag 3360

aacagttttc attcacttac taatccactc tgtgaggaaa agcttttgga gaaggaaagc 3420

ttggatttag cccgaaaaga tagggaatca aatcatgatc ttagtcatgg ttgctctgac 3480

gaggaacatg atcatctaaa tttagacaag gcacatgaca gttgtgcctt acaagaatgt 3540

tgttattctg ttcaaggcaa taaaactgat gtatcagaaa ctggaatcca ggaagctgct 3600

cattgtgaca gcatcaatca aacatgccaa actgcaattt caggatcaat gacatgcgga 3660

aataataaga gtctggactc tctaagcatc catggttgtc attcacatga tagtccactc 3720

cacaaggaga gcaacttgga gcagaaaagc ttggatgttg ctggagaagg tataaaatca 3780

cctcatgctg tcggtcaagg ctgttcggac aaggagcaca atcactcgca tccagaaaag 3840

gcgtatgaca gttgtgcaac agacgattgt tgtttttcag ttcaagtcca tggcattgac 3900

gacgtatcaa gaagtgaaat tcaagaaact gctcattgtg acagcacaaa acagagcacg 3960

gtcatcccca gcagctgcga acatgaacca aaagatcagg taaatcactg tggatctcac 4020

tctaaaagta ttccaactga tgaagaacta gccaagctgg ttagaagatg ctgcaaatac 4080

aaaccatgcc acgatgtccg ctctggctgc aggaagcatg ctgcagaatg tggtccaacc 4140

gttcgatcaa ccatcaatat cttacgggac aaccatcatc atcatctaga ctgcagtggt 4200

cgtaaggttt gttcgctgtt ggagaagaga cacattggtg gatgctgtga cagcttcaga 4260

aaagaatgtt gtgccaagaa caatcacctt ggagcaagtt ttggaggagg tttatcagaa 4320

attgtcatag agtag 4335

<210> 4

<211> 1444

<212> PRT

<213> NtHMA4基因氨基酸

<400> 4

Met Val Glu Ser Glu Lys Met Asn Asp Thr Lys Asn Leu Ser Lys Ser

1 5 10 15

Tyr Phe Asp Val Leu Gly Ile Cys Cys Thr Ser Glu Val Val Leu Val

20 25 30

Glu Lys Ile Leu Lys Asn Leu Glu Gly Val Lys Glu Val Ser Val Ile

35 40 45

Val Thr Ile Lys Thr Val Ile Val Ile His Asp Ser Leu Leu Ile Ser

50 55 60

Gln Gln Gln Ile Val Lys Ala Leu Asn Gln Ala Arg Leu Glu Ala Ser

65 70 75 80

Ile Arg Val Lys Gly Glu Lys Asn Tyr Gln Lys Lys Trp Pro Ser Pro

85 90 95

Phe Ala Ile Gly Ser Gly Ile Leu Leu Gly Leu Ser Phe Leu Lys Tyr

100 105 110

Phe Phe Ala Pro Phe Gln Trp Leu Ala Leu Ala Ala Val Ala Val Gly

115 120 125

Ile Pro Pro Ile Ile Phe Arg Gly Val Ala Ala Val Arg Asn Leu Thr

130 135 140

Leu Asp Ile Asn Ile Leu Val Leu Ile Ala Val Thr Gly Ser Ile Val

145 150 155 160

Leu His Asp Tyr Trp Glu Ala Gly Thr Ile Val Phe Leu Phe Thr Ile

165 170 175

Ala Glu Trp Leu Glu Ser Arg Ala Ser His Lys Ala Thr Ala Ala Met

180 185 190

Ser Ser Leu Val Asn Ile Val Pro Pro Thr Ala Val Leu Ala Glu Ser

195 200 205

Gly Glu Val Val Asn Val Asp Glu Val Lys Leu Asn Ser Ile Leu Ala

210 215 220

Val Lys Ala Gly Glu Thr Ile Pro Ile Asp Gly Val Val Met Glu Gly

225 230 235 240

Glu Cys Asp Val Asp Glu Lys Thr Leu Thr Gly Glu Ser Phe Pro Val

245 250 255

Ser Lys Gln Ile Asp Ser Thr Val Trp Ala Gly Thr Thr Asn Leu Asn

260 265 270

Gly Tyr Ile Ser Val Lys Thr Thr Ala Leu Ala Glu Asp Cys Ala Val

275 280 285

Ala Arg Met Ala Gln Leu Val Glu Asp Ala Gln Asn Lys Lys Ser Lys

290 295 300

Thr Gln Arg Tyr Ile Asp Lys Cys Ala Lys Tyr Tyr Thr Pro Ala Ile

305 310 315 320

Val Ala Ile Ser Ala Ser Leu Ala Ile Val Pro Thr Ala Leu Arg Val

325 330 335

His Asn Arg Asn Glu Trp Tyr Arg Leu Ala Leu Val Thr Leu Val Ser

340 345 350

Ala Cys Pro Cys Ala Leu Val Leu Ser Thr Pro Val Ala Met Cys Cys

355 360 365

Ala Leu Ser Lys Ala Ala Thr Ser Gly Leu Leu Phe Lys Gly Ala Glu

370 375 380

Tyr Leu Glu Thr Leu Ala Lys Ile Lys Ile Met Ala Phe Asp Lys Thr

385 390 395 400

Gly Thr Ile Thr Arg Gly Glu Phe Met Val Thr Glu Phe Lys Ser Leu

405 410 415

Val Asp Gly Leu Gly Leu Asn Thr Leu Leu Tyr Trp Val Ser Ser Ile

420 425 430

Glu Ser Lys Ser Gly His Pro Met Ala Ala Ala Leu Val Asp Tyr Ala

435 440 445

Gln Ser Asn Ser Val Glu Pro Lys Pro Asp Arg Val Glu Gln Phe Gln

450 455 460

Asn Phe Pro Gly Glu Gly Ile Phe Gly Arg Ile Asp Gly Met Glu Ile

465 470 475 480

Tyr Val Gly Asn Arg Lys Ile Ser Ser Arg Ala Gly Cys Thr Thr Val

485 490 495

Pro Glu Ile Glu Gly Asp Ser Phe Gln Gly Lys Ser Val Gly Tyr Ile

500 505 510

Phe Leu Gly Ser Ser Pro Ala Gly Ile Phe Gly Leu Ser Asp Val Cys

515 520 525

Arg Ile Gly Val Lys Glu Ala Met Arg Glu Leu Lys Gln Met Gly Ile

530 535 540

Lys Thr Ala Met Leu Thr Gly Asp Cys Tyr Ala Ala Ala Asn His Val

545 550 555 560

Gln Asp Gln Leu Gly Gly Ala Met Asp Glu Phe Gln Ala Glu Leu Leu

565 570 575

Pro Glu Asp Lys Ala Thr Ile Ile Lys Gly Phe Gln Lys Glu Ala Pro

580 585 590

Thr Ala Met Ile Gly Asp Gly Leu Asn Asp Ala Pro Ala Leu Ala Thr

595 600 605

Ala Asp Ile Gly Ile Ser Met Gly Ile Ser Gly Ser Ala Leu Ala Lys

610 615 620

Glu Thr Gly His Val Ile Leu Met Thr Asn Asp Ile Gly Arg Ile Pro

625 630 635 640

Lys Ala Ala Arg Leu Ala Arg Arg Val Arg Arg Lys Ile Val Glu Asn

645 650 655

Met Ile Ile Ser Val Val Thr Lys Ala Ala Ile Val Ala Leu Ala Ile

660 665 670

Ala Gly Tyr Pro Leu Val Trp Ala Ala Val Leu Ala Asp Thr Gly Thr

675 680 685

Cys Leu Leu Val Ile Leu Asn Ser Met Leu Leu Leu Arg Val Gly Thr

690 695 700

His Arg His Gly Lys Lys Cys Cys Arg Ser Ala Thr Pro Ser His Ala

705 710 715 720

Pro Asn His Lys Asp Lys Ala Ser Cys Cys Lys Ser Glu Asn Ala Pro

725 730 735

Gln Leu Cys Cys Ser Asp Ile Glu Ser Gln Lys Lys Cys Thr Ser Gln

740 745 750

Ser Cys Ser Ser Glu Val Cys Val Pro Arg Cys Gln Pro Val Ser Ser

755 760 765

Gly Ser Lys Ser Cys Gly Asn Asn Gln Cys Pro Asp Ser Val Glu Asn

770 775 780

Ser Gly Phe His Ser His Pro Arg Pro Gln Cys Cys Ser Ser Lys Met

785 790 795 800

Ala Ser Lys Ala Cys Gln Ser Ala Val Ser Glu Ser Lys Ser Cys Gly

805 810 815

Asn Asn Gln Cys Pro Asp Ser Val Glu Asn Ser Gly Phe His Ser His

820 825 830

Pro Arg Pro Gln Cys Cys Ser Ser Lys Met Ala Ser Lys Ala Cys Gln

835 840 845

Ser Ala Val Ser Glu Ser Lys Ser Cys Gly Asn Asn Gln Cys Pro Asp

850 855 860

Ser Val Glu Asn Ser Gly Phe His Ser His Pro Arg Pro Gln Cys Cys

865 870 875 880

Ser Leu Lys Met Ala Ser Lys Ala Cys Gln Ser Ala Val Ser Glu Ser

885 890 895

Lys Ser Cys Gly Asn Asn Gln Cys Pro Asp Ser Val Glu Asn Ser Gly

900 905 910

Phe His Ser His Pro Arg Pro Gln Cys Cys Ser Ser Lys Met Ala Ala

915 920 925

Lys Ala Cys Gln Ser Ala Val Ser Glu Ser Lys Ser Cys Gly Asn Asn

930 935 940

Asn Cys Ser Glu Ser Ile Tyr Lys Ser Ser Cys His Ser Leu Thr Ser

945 950 955 960

Ser Leu Val Cys Ser Ser Lys Met Ser Ala Pro Gln Cys His Ser Ala

965 970 975

Thr Ser Ser Ser Lys Ser Cys Gly Ser Thr Lys Cys Ser Asn Phe Ser

980 985 990

Asp Lys Lys Cys Cys Gln Tyr Asp Lys Ile Pro Gln Thr Cys Ser Thr

995 1000 1005

Lys Lys Ser Ala Pro Gly Cys Gln Ser Ala Val Ser Gly Ser Lys

1010 1015 1020

Ser Cys Gly Asp Ser Lys Cys Ser Asp Ser Lys Asp Asn Ser Ser

1025 1030 1035

His Pro Ser His Pro Asp His Gln Ile Cys Thr Ser Lys Leu Cys

1040 1045 1050

Ala Pro Gln Ser Gln Ser Ala Thr Ser Ser Ser Arg Thr Cys Gly

1055 1060 1065

Asn Met Lys Cys Ser Asp Thr Asn Ser Lys Asn Ser Cys Tyr Ser

1070 1075 1080

His Thr Asn Ser Glu Ser Cys Ser Ser Lys Met Ser Gly Pro Ala

1085 1090 1095

Cys Lys Thr Ala Asn Ser Gly Ser Arg Leu Cys Gly Asn Lys Lys

1100 1105 1110

Cys Leu Asp Ser Ala Asn Glu Asn Ser Phe His Ser Leu Thr Asn

1115 1120 1125

Pro Leu Cys Glu Glu Lys Leu Leu Glu Lys Glu Ser Leu Asp Leu

1130 1135 1140

Ala Arg Lys Asp Arg Glu Ser Asn His Asp Leu Ser His Gly Cys

1145 1150 1155

Ser Asp Glu Glu His Asp His Leu Asn Leu Asp Lys Ala His Asp

1160 1165 1170

Ser Cys Ala Leu Gln Glu Cys Cys Tyr Ser Val Gln Gly Asn Lys

1175 1180 1185

Thr Asp Val Ser Glu Thr Gly Ile Gln Glu Ala Ala His Cys Asp

1190 1195 1200

Ser Ile Asn Gln Thr Cys Gln Thr Ala Ile Ser Gly Ser Met Thr

1205 1210 1215

Cys Gly Asn Asn Lys Ser Leu Asp Ser Leu Ser Ile His Gly Cys

1220 1225 1230

His Ser His Asp Ser Pro Leu His Lys Glu Ser Asn Leu Glu Gln

1235 1240 1245

Lys Ser Leu Asp Val Ala Gly Glu Gly Ile Lys Ser Pro His Ala

1250 1255 1260

Val Gly Gln Gly Cys Ser Asp Lys Glu His Asn His Ser His Pro

1265 1270 1275

Glu Lys Ala Tyr Asp Ser Cys Ala Thr Asp Asp Cys Cys Phe Ser

1280 1285 1290

Val Gln Val His Gly Ile Asp Asp Val Ser Arg Ser Glu Ile Gln

1295 1300 1305

Glu Thr Ala His Cys Asp Ser Thr Lys Gln Ser Thr Val Ile Pro

1310 1315 1320

Ser Ser Cys Glu His Glu Pro Lys Asp Gln Val Asn His Cys Gly

1325 1330 1335

Ser His Ser Lys Ser Ile Pro Thr Asp Glu Glu Leu Ala Lys Leu

1340 1345 1350

Val Arg Arg Cys Cys Lys Tyr Lys Pro Cys His Asp Val Arg Ser

1355 1360 1365

Gly Cys Arg Lys His Ala Ala Glu Cys Gly Pro Thr Val Arg Ser

1370 1375 1380

Thr Ile Asn Ile Leu Arg Asp Asn His His His His Leu Asp Cys

1385 1390 1395

Ser Gly Arg Lys Val Cys Ser Leu Leu Glu Lys Arg His Ile Gly

1400 1405 1410

Gly Cys Cys Asp Ser Phe Arg Lys Glu Cys Cys Ala Lys Asn Asn

1415 1420 1425

His Leu Gly Ala Ser Phe Gly Gly Gly Leu Ser Glu Ile Val Ile

1430 1435 1440

Glu

<210> 5

<211> 30

<212> DNA

<213> HMA4-F

<400> 5

tagagtgtag aggaaaaata gaaagaagag 30

<210> 6

<211> 30

<212> DNA

<213> HMA4-R

<400> 6

ataagctgag agcttaagaa aaaagaaact 30

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