本发明属于遗传工程技术领域,具体涉及一种NtHMA2基因突变体与应用。
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NtHMA2基因的EMS突变体;第二目的在于提供所述的NtHMA2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NtHMA2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NtHMA2基因序列520位的C突变为T,形成终止密码子,使该基因提前终止。
所述的NtHMA2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NtHMA2基因TILLING分析引物,HMA2-F:
AATATAGAGATGGCCAATGAAGGT和HMA2-R: CTAAAATTTATATGACCAAGTACTT,扩增长度为1400bp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520位的C突变为T,形成终止密码子,使该基因提前终止。
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40%左右,锌、铁、铅、铬、镍等含量降低30%左右,砷含量降低10%左右。
本发明的第二目的是这样实现的,NtHMA2基因突变体可以使烟叶镉含量降低30~50%,锌、铁、铅、铬、镍等含量降低20~40%左右,砷含量降低5~15%左右。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NtHMA2突变体与云烟87叶片镉含量对比示意图;
图3为本发明NtHMA2突变体与云烟87叶片锌含量对比示意图;
图4为本发明NtHMA2突变体与云烟87叶片铁含量对比示意图;
图5为本发明NtHMA2突变体与云烟87叶片砷含量对比示意图;
图6为本发明NtHMA2突变体与云烟87叶片铬含量对比示意图;
图7为本发明NtHMA2突变体与云烟87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。
本发明所述的NtHMA2基因突变体,与野生型烟草的NtHMA2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NtHMA2基因序列520位的C突变为T,形成终止密码子,使该基因提前终止。
NtHMA2基因编码的氨基酸序列如SEQ ID No:2所示;
NtHMA2基因突变体的核苷酸序列如SEQ ID No:3所示;
NtHMA2基因突变体编码的氨基酸序列如SEQ ID No:4所示;
所述的NtHMA2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NtHMA2基因TILLING分析引物,
HMA2-F:AATATAGAGATGGCCAATGAAGGT和
HMA2-R: CTAAAATTTATATGACCAAGTACTT,扩增长度为1400bp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520位的C突变为T,形成终止密码子,使该基因提前终止。
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40%左右,锌、铁、铅、铬、镍等含量降低30%左右,砷含量降低10%左右。
本发明所述的应用为NtHMA2基因突变体可以使烟叶镉含量降低30~50%左右,锌、铁、铅、铬、镍等含量降低20~40%左右,砷含量降低5~15%左右。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NtHMA2突变体的制备方法, 未突变NtHMA2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520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NtHMA2基因TILLING分析引物,
HMA2-F:AATATAGAGATGGCCAATGAAGGT和
HMA2-R:CTAAAATTTATATGACCAAGTACTT,扩增长度为1400bp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 HMA2-F primer(10µM) ,0.16µl HMA2-R 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。
(11)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NtHMA2基因CDS的520位的C突变为T,形成终止密码子,使该基因提前终止。
(12)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。
NtHMA2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40%左右,锌、铁、铅、铬、镍等含量降低30%左右,砷含量降低10%左右(表1,图2~图7)。
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NtHMA2突变体与应用
<130> 2017
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 4212
<212> DNA
<213> NtHMA2基因核苷酸
<400> 1
atggtggaaa gtgaaaaaat gaatgaaaca aagaagttga gcaagagcta ttttgatgtt 60
ttgggaattt gctgtacttc agaagttgtt ctagttgaaa aaattctcaa gaatcttgaa 120
ggggttaaag aggtttcagt aattgtcaca acaaagactg tcattgttat tcatgattct 180
cttctcattt ctccgcaaca aattgttaaa gcattgaatc aagcaagatt agaagcaagc 240
ataagagtga aaggagagaa aaactaccaa aagaaatggc caagtccatt tgcaattggc 300
agtggaatat tgcttggact ctcatttttg aagtactttt ttgcaccttt ccaatggtta 360
gcacttgcag ctgttgcagt tgggattcct ccaattattt ttagaggtgt ggctgccgtg 420
cgaaacctca ctcttgacat caacattctt gttttaatag cagtggctgg atcaattgtt 480
ttacacgatt attgggaagc tggtactatt gtcttcttat tcgccattgc agaatggcta 540
gagtcaaggg caagtcacaa ggctaccgct gctatgtcat cactggtcaa tatagtccct 600
ccaacagcag ttttagctga aagcggagaa gtcgtaaatg ttgatgaagt caaggtgaat 660
agcattcttg ctgtgaaagc tggtgaaact atacctattg atggagttgt agtggaaggg 720
gaatgtgacg tggacgagaa aacactgaca ggcgagtcgt ttccagtttc taagcaaaga 780
gattcaacgg tctgggctgg cactacaaat ctaaatggct atatcagtgt taagactacg 840
gctttggctg aagattgtgc ggtggctagg atggcacagc ttgtcgaaga tgctcagaac 900
aagaaatcaa aaacccaaag atacatcgac aagtgtgcta aatattatac accagcaatt 960
gtggctatat cagcttcttt ggcaattgtt cctactgcat taagagttca caatcgaaat 1020
gaatggtatc gcttggcttt ggtcacattg gtgagtgcat gtccgtgtgc acttgttcta 1080
tctacaccag ttgccatgtg ttgcgcactt tcaaaagcag caacgtccgg tcttctgttt 1140
aaaggagcag agtaccttga gactctagct aaaatcaaaa tcatggcttt tgacaaaaca 1200
gggactataa ctaaaggaga atttatggtg accgagttca agtctctgat tgatggtttt 1260
agtctcaata cactgcttta ctgggtttca agcattgaga gcaagtcagg tcatccgatg 1320
gcagccgctc tggtggacta tgcacaatca aattccgttg agccaaagcc tgatagagtt 1380
gagcagtttc aaaattttcc tggtgaaggg atatttggaa gaattgatgg aatggaaatc 1440
tatgtcggga ataggaaaat ttcttcaaga gctggatgta ccacagtacc agaaatagag 1500
ggtgatagtt tcaaaggaaa gtctgttgga tacatatttt tgggatcatc tccagctgga 1560
attttcagtc tttccgatgt ttgtcgaatt ggtgtaaaag aagcaatgag agaactgaag 1620
cagatgggta tcaaaaccgc gatgcttact ggtgattgtt atgcagctgc caaccatgtg 1680
caggatcagt taggtggagc tttggatgaa tttcaagcag aactcctacc agaggacaag 1740
gcaacaatca tcaagggttt tcagaaggaa gctccaacag cgatgatagg cgacggcctt 1800
aatgatgctc ctgcattagc aacagctgac attggcatct caatgggcat ctctgggtca 1860
gctctcgcta aagaaacagg ccatgttata ctaatgacaa atgacatcgg aagaataccg 1920
aaagctgcac gtcttgctag aagagttcga aggaagattg ttgagaatat gattatatca 1980
gtcgttacaa aggctgccat agttgcattg gcaatagcag gttatccatt ggtttgggct 2040
gctgtcctcg cagatactgg gacatgcttg ctagtgattt tgaacagcat gctacttcta 2100
cgaggaggca cacgcagaca tgggaaaaaa tgttggagat cttctactcc ttcgcatgct 2160
ccccaccaca aagacaaagc ttcatgttgc aagtcggaaa atgctcccca gctgtgttgc 2220
tctgatattg agtcacaaaa gaaatgtaca agtcaatcat gctcgtccga ggtgtgtgtt 2280
ccaagatgtc aacctgtctc ctcaggatca aagtcatgtg gaaataatca gtgcccagac 2340
tccattgaaa atagtggttt tcattctcat ccccgtcctc aatgctgctc gtcgaagatg 2400
gctgctaaag catgccaatc tgcagtttca gaatcaaagt catgcggaaa taatcagtgc 2460
ccagactccg ttgaaaatag tggttttcat tctcatcccc gtcctgaatg ctgctcgtcg 2520
aagatggctg ctaaagcgtg ccaatctgca gtttcagaat caaagtcatg tggaaataat 2580
cagtgcccag actccgttga aaatagtggt tttcattctc atccccgtcc tcaatgctgt 2640
tcatcgaaga tggctgctaa agcaggccaa tctgcacttt cagaatcaaa gtcatgtgga 2700
aataacaatt gctcagactc cattcacaag agtaattgtc attctttaac taactctcta 2760
gtatgttctt ccaagatgtc tgctccacaa tgtcattctg ctacttcaag caacaaatca 2820
tgtggaagta ccaagtgctc cgacttcagt gacaaaaaat gttgtcaatc cgacaaaatt 2880
cctcaaacgt gctctaccaa gaagtctgct ccaggatgtc aatctgcagt ttctgggtct 2940
aaatcatgtg gaaatagcaa gtgttcagac tcaaaagaca atagtagcca tccttcacat 3000
cccgatcatc aaacatgcat gtctaagttg tgtgctccac aaagccaatc tgcaacttca 3060
agctccagga catgtggaaa tacaaagtgc tcggacacca atagcaagaa ttcttgttat 3120
tcacaaacca actctgaatc atgctcttca aagatgtctg gtccatcatg caaaactgct 3180
aattcaggtt caaggtcatg cagaaataag aagtgccagg actctgcaac cgagaacagt 3240
tttcattcac cacttactaa tccactcagt ggggaaaagc tttcggagca gaaaagcttg 3300
gatttagtcc gaaaagataa ggaatcaagt catgatcttc gtcatggctg ctctgacgag 3360
gaacatgatc atacaaattt agacaaggca tatgacagtt gtgccttaca agaatgttgt 3420
tattcggttc aaggcaataa aactgatgta tcagaaactg gaatccagga aactgctcat 3480
tgtgacagca ccaatcaaac atgccaaact gcaagttcag gatcgatgac atgcggaaat 3540
gataagatcc tggactctct aagcatccat ggttgtcatt cgcatgataa tccactccac 3600
gaggagaaca acttggagca gaaaatcttg gatgttgttg gagaaggtat aaaatcacct 3660
catgctgtcg gtcatggctg ttcggacaag gaacacgatc actcacatcc agaaaaggca 3720
tatgacagtt gtgcaacaga tgattgttgt ttttcagttc aagtccatgg cattgacgac 3780
gtatcaaaaa gtgaaattca agaaactgct cattgtgaca gcacaaagca gagcatggtc 3840
atctccagca gctgcaaaca tgaaccaaaa gatcaggtaa atcactgtgg acttcactct 3900
aaaactactc caactgatga agaactagcc aagctggtta gaagatgctg caaatacaaa 3960
ccatgccacg acgtccgttc tggctgcagg aagcatgctg cagaatgtgg tccaaccgtt 4020
cgatcaacca tcaatatctt acgggacaac catcatcatt acctagactg cagtggtcgt 4080
aaggtttgtt cgctgttgga gaagagacac atcggtggat gctgtgacag cttcagaaaa 4140
gaatgttgtg ccaagaaaaa acaccttgga gcaagttttg gaggaggttt atcagaaatt 4200
gtcatagagt ag 4212
<210> 2
<211> 1403
<212> PRT
<213> NtHMA2基因氨基酸
<400> 2
Met Val Glu Ser Glu Lys Met Asn Glu Thr Lys Lys 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
Pro 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 Ala Gly Ser Ile Val
145 150 155 160
Leu His Asp Tyr Trp Glu Ala Gly Thr Ile Val Phe Leu Phe Ala 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 Val Asn Ser Ile Leu Ala
210 215 220
Val Lys Ala Gly Glu Thr Ile Pro Ile Asp Gly Val Val Val 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 Arg 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 Lys Gly Glu Phe Met Val Thr Glu Phe Lys Ser Leu
405 410 415
Ile Asp Gly Phe Ser 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 Lys Gly Lys Ser Val Gly Tyr Ile
500 505 510
Phe Leu Gly Ser Ser Pro Ala Gly Ile Phe Ser 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 Leu 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 Gly Gly Thr
690 695 700
Arg Arg His Gly Lys Lys Cys Trp Arg Ser Ser Thr Pro Ser His Ala
705 710 715 720
Pro His 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 Ile 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 Ala 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 Glu Cys Cys Ser Ser Lys Met Ala Ala 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 Ser Lys Met Ala Ala Lys Ala Gly Gln Ser Ala Leu Ser Glu Ser
885 890 895
Lys Ser Cys Gly Asn Asn Asn Cys Ser Asp Ser Ile His Lys Ser Asn
900 905 910
Cys His Ser Leu Thr Asn Ser Leu Val Cys Ser Ser Lys Met Ser Ala
915 920 925
Pro Gln Cys His Ser Ala Thr Ser Ser Asn Lys Ser Cys Gly Ser Thr
930 935 940
Lys Cys Ser Asp Phe Ser Asp Lys Lys Cys Cys Gln Ser Asp Lys Ile
945 950 955 960
Pro Gln Thr Cys Ser Thr Lys Lys Ser Ala Pro Gly Cys Gln Ser Ala
965 970 975
Val Ser Gly Ser Lys Ser Cys Gly Asn Ser Lys Cys Ser Asp Ser Lys
980 985 990
Asp Asn Ser Ser His Pro Ser His Pro Asp His Gln Thr Cys Met Ser
995 1000 1005
Lys Leu Cys Ala Pro Gln Ser Gln Ser Ala Thr Ser Ser Ser Arg
1010 1015 1020
Thr Cys Gly Asn Thr Lys Cys Ser Asp Thr Asn Ser Lys Asn Ser
1025 1030 1035
Cys Tyr Ser Gln Thr Asn Ser Glu Ser Cys Ser Ser Lys Met Ser
1040 1045 1050
Gly Pro Ser Cys Lys Thr Ala Asn Ser Gly Ser Arg Ser Cys Arg
1055 1060 1065
Asn Lys Lys Cys Gln Asp Ser Ala Thr Glu Asn Ser Phe His Ser
1070 1075 1080
Pro Leu Thr Asn Pro Leu Ser Gly Glu Lys Leu Ser Glu Gln Lys
1085 1090 1095
Ser Leu Asp Leu Val Arg Lys Asp Lys Glu Ser Ser His Asp Leu
1100 1105 1110
Arg His Gly Cys Ser Asp Glu Glu His Asp His Thr Asn Leu Asp
1115 1120 1125
Lys Ala Tyr Asp Ser Cys Ala Leu Gln Glu Cys Cys Tyr Ser Val
1130 1135 1140
Gln Gly Asn Lys Thr Asp Val Ser Glu Thr Gly Ile Gln Glu Thr
1145 1150 1155
Ala His Cys Asp Ser Thr Asn Gln Thr Cys Gln Thr Ala Ser Ser
1160 1165 1170
Gly Ser Met Thr Cys Gly Asn Asp Lys Ile Leu Asp Ser Leu Ser
1175 1180 1185
Ile His Gly Cys His Ser His Asp Asn Pro Leu His Glu Glu Asn
1190 1195 1200
Asn Leu Glu Gln Lys Ile Leu Asp Val Val Gly Glu Gly Ile Lys
1205 1210 1215
Ser Pro His Ala Val Gly His Gly Cys Ser Asp Lys Glu His Asp
1220 1225 1230
His Ser His Pro Glu Lys Ala Tyr Asp Ser Cys Ala Thr Asp Asp
1235 1240 1245
Cys Cys Phe Ser Val Gln Val His Gly Ile Asp Asp Val Ser Lys
1250 1255 1260
Ser Glu Ile Gln Glu Thr Ala His Cys Asp Ser Thr Lys Gln Ser
1265 1270 1275
Met Val Ile Ser Ser Ser Cys Lys His Glu Pro Lys Asp Gln Val
1280 1285 1290
Asn His Cys Gly Leu His Ser Lys Thr Thr Pro Thr Asp Glu Glu
1295 1300 1305
Leu Ala Lys Leu Val Arg Arg Cys Cys Lys Tyr Lys Pro Cys His
1310 1315 1320
Asp Val Arg Ser Gly Cys Arg Lys His Ala Ala Glu Cys Gly Pro
1325 1330 1335
Thr Val Arg Ser Thr Ile Asn Ile Leu Arg Asp Asn His His His
1340 1345 1350
Tyr Leu Asp Cys Ser Gly Arg Lys Val Cys Ser Leu Leu Glu Lys
1355 1360 1365
Arg His Ile Gly Gly Cys Cys Asp Ser Phe Arg Lys Glu Cys Cys
1370 1375 1380
Ala Lys Lys Lys His Leu Gly Ala Ser Phe Gly Gly Gly Leu Ser
1385 1390 1395
Glu Ile Val Ile Glu
1400
<210> 3
<211> 4212
<212> DNA
<213> NtHMA2基因突变体核苷酸
<400> 3
atggtggaaa gtgaaaaaat gaatgaaaca aagaagttga gcaagagcta ttttgatgtt 60
ttgggaattt gctgtacttc agaagttgtt ctagttgaaa aaattctcaa gaatcttgaa 120
ggggttaaag aggtttcagt aattgtcaca acaaagactg tcattgttat tcatgattct 180
cttctcattt ctccgcaaca aattgttaaa gcattgaatc aagcaagatt agaagcaagc 240
ataagagtga aaggagagaa aaactaccaa aagaaatggc caagtccatt tgcaattggc 300
agtggaatat tgcttggact ctcatttttg aagtactttt ttgcaccttt ccaatggtta 360
gcacttgcag ctgttgcagt tgggattcct ccaattattt ttagaggtgt ggctgccgtg 420
cgaaacctca ctcttgacat caacattctt gttttaatag cagtggctgg atcaattgtt 480
ttacacgatt attgggaagc tggtactatt gtcttcttat tcgccattgc agaatggcta 540
gagtcaaggg caagtcacaa ggctaccgct gctatgtcat cactggtcaa tatagtccct 600
ccaacagcag ttttagctga aagcggagaa gtcgtaaatg ttgatgaagt caaggtgaat 660
agcattcttg ctgtgaaagc tggtgaaact atacctattg atggagttgt agtggaaggg 720
gaatgtgacg tggacgagaa aacactgaca ggcgagtcgt ttccagtttc taagcaaaga 780
gattcaacgg tctgggctgg cactacaaat ctaaatggct atatcagtgt taagactacg 840
gctttggctg aagattgtgc ggtggctagg atggcatagc ttgtcgaaga tgctcagaac 900
aagaaatcaa aaacccaaag atacatcgac aagtgtgcta aatattatac accagcaatt 960
gtggctatat cagcttcttt ggcaattgtt cctactgcat taagagttca caatcgaaat 1020
gaatggtatc gcttggcttt ggtcacattg gtgagtgcat gtccgtgtgc acttgttcta 1080
tctacaccag ttgccatgtg ttgcgcactt tcaaaagcag caacgtccgg tcttctgttt 1140
aaaggagcag agtaccttga gactctagct aaaatcaaaa tcatggcttt tgacaaaaca 1200
gggactataa ctaaaggaga atttatggtg accgagttca agtctctgat tgatggtttt 1260
agtctcaata cactgcttta ctgggtttca agcattgaga gcaagtcagg tcatccgatg 1320
gcagccgctc tggtggacta tgcacaatca aattccgttg agccaaagcc tgatagagtt 1380
gagcagtttc aaaattttcc tggtgaaggg atatttggaa gaattgatgg aatggaaatc 1440
tatgtcggga ataggaaaat ttcttcaaga gctggatgta ccacagtacc agaaatagag 1500
ggtgatagtt tcaaaggaaa gtctgttgga tacatatttt tgggatcatc tccagctgga 1560
attttcagtc tttccgatgt ttgtcgaatt ggtgtaaaag aagcaatgag agaactgaag 1620
cagatgggta tcaaaaccgc gatgcttact ggtgattgtt atgcagctgc caaccatgtg 1680
caggatcagt taggtggagc tttggatgaa tttcaagcag aactcctacc agaggacaag 1740
gcaacaatca tcaagggttt tcagaaggaa gctccaacag cgatgatagg cgacggcctt 1800
aatgatgctc ctgcattagc aacagctgac attggcatct caatgggcat ctctgggtca 1860
gctctcgcta aagaaacagg ccatgttata ctaatgacaa atgacatcgg aagaataccg 1920
aaagctgcac gtcttgctag aagagttcga aggaagattg ttgagaatat gattatatca 1980
gtcgttacaa aggctgccat agttgcattg gcaatagcag gttatccatt ggtttgggct 2040
gctgtcctcg cagatactgg gacatgcttg ctagtgattt tgaacagcat gctacttcta 2100
cgaggaggca cacgcagaca tgggaaaaaa tgttggagat cttctactcc ttcgcatgct 2160
ccccaccaca aagacaaagc ttcatgttgc aagtcggaaa atgctcccca gctgtgttgc 2220
tctgatattg agtcacaaaa gaaatgtaca agtcaatcat gctcgtccga ggtgtgtgtt 2280
ccaagatgtc aacctgtctc ctcaggatca aagtcatgtg gaaataatca gtgcccagac 2340
tccattgaaa atagtggttt tcattctcat ccccgtcctc aatgctgctc gtcgaagatg 2400
gctgctaaag catgccaatc tgcagtttca gaatcaaagt catgcggaaa taatcagtgc 2460
ccagactccg ttgaaaatag tggttttcat tctcatcccc gtcctgaatg ctgctcgtcg 2520
aagatggctg ctaaagcgtg ccaatctgca gtttcagaat caaagtcatg tggaaataat 2580
cagtgcccag actccgttga aaatagtggt tttcattctc atccccgtcc tcaatgctgt 2640
tcatcgaaga tggctgctaa agcaggccaa tctgcacttt cagaatcaaa gtcatgtgga 2700
aataacaatt gctcagactc cattcacaag agtaattgtc attctttaac taactctcta 2760
gtatgttctt ccaagatgtc tgctccacaa tgtcattctg ctacttcaag caacaaatca 2820
tgtggaagta ccaagtgctc cgacttcagt gacaaaaaat gttgtcaatc cgacaaaatt 2880
cctcaaacgt gctctaccaa gaagtctgct ccaggatgtc aatctgcagt ttctgggtct 2940
aaatcatgtg gaaatagcaa gtgttcagac tcaaaagaca atagtagcca tccttcacat 3000
cccgatcatc aaacatgcat gtctaagttg tgtgctccac aaagccaatc tgcaacttca 3060
agctccagga catgtggaaa tacaaagtgc tcggacacca atagcaagaa ttcttgttat 3120
tcacaaacca actctgaatc atgctcttca aagatgtctg gtccatcatg caaaactgct 3180
aattcaggtt caaggtcatg cagaaataag aagtgccagg actctgcaac cgagaacagt 3240
tttcattcac cacttactaa tccactcagt ggggaaaagc tttcggagca gaaaagcttg 3300
gatttagtcc gaaaagataa ggaatcaagt catgatcttc gtcatggctg ctctgacgag 3360
gaacatgatc atacaaattt agacaaggca tatgacagtt gtgccttaca agaatgttgt 3420
tattcggttc aaggcaataa aactgatgta tcagaaactg gaatccagga aactgctcat 3480
tgtgacagca ccaatcaaac atgccaaact gcaagttcag gatcgatgac atgcggaaat 3540
gataagatcc tggactctct aagcatccat ggttgtcatt cgcatgataa tccactccac 3600
gaggagaaca acttggagca gaaaatcttg gatgttgttg gagaaggtat aaaatcacct 3660
catgctgtcg gtcatggctg ttcggacaag gaacacgatc actcacatcc agaaaaggca 3720
tatgacagtt gtgcaacaga tgattgttgt ttttcagttc aagtccatgg cattgacgac 3780
gtatcaaaaa gtgaaattca agaaactgct cattgtgaca gcacaaagca gagcatggtc 3840
atctccagca gctgcaaaca tgaaccaaaa gatcaggtaa atcactgtgg acttcactct 3900
aaaactactc caactgatga agaactagcc aagctggtta gaagatgctg caaatacaaa 3960
ccatgccacg acgtccgttc tggctgcagg aagcatgctg cagaatgtgg tccaaccgtt 4020
cgatcaacca tcaatatctt acgggacaac catcatcatt acctagactg cagtggtcgt 4080
aaggtttgtt cgctgttgga gaagagacac atcggtggat gctgtgacag cttcagaaaa 4140
gaatgttgtg ccaagaaaaa acaccttgga gcaagttttg gaggaggttt atcagaaatt 4200
gtcatagagt ag 4212
<210> 4
<211> 292
<212> PRT
<213> NtHMA2基因突变体氨基酸
<400> 4
Met Val Glu Ser Glu Lys Met Asn Glu Thr Lys Lys 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
Pro 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 Ala Gly Ser Ile Val
145 150 155 160
Leu His Asp Tyr Trp Glu Ala Gly Thr Ile Val Phe Leu Phe Ala 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 Val Asn Ser Ile Leu Ala
210 215 220
Val Lys Ala Gly Glu Thr Ile Pro Ile Asp Gly Val Val Val 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 Arg 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
290
<210> 5
<211> 24
<212> DNA
<213> HMA2-F
<400> 5
aatatagaga tggccaatga aggt 24
<210> 6
<211> 25
<212> DNA
<213> HMA2-R
<400> 6
ctaaaattta tatgaccaag tactt 25