CN116286705B - 一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2及其应用 - Google Patents
一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2及其应用 Download PDFInfo
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- CN116286705B CN116286705B CN202310052969.2A CN202310052969A CN116286705B CN 116286705 B CN116286705 B CN 116286705B CN 202310052969 A CN202310052969 A CN 202310052969A CN 116286705 B CN116286705 B CN 116286705B
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Abstract
本发明公开了一种本发明涉及基因功能鉴定技术领域，公开了一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2及其应用。本发明提供了构树BpHMT2基因的序列，并提供了其在提高植物耐硒能力以及提高植物对硒吸收和积累能力中的应用。本发明在提高植物总硒含量的同时增加了有机硒成分硒代蛋氨酸的生成，可将其作为靶基因，通过基因工程手段提高构树作为饲料原料的营养价值，指导构树的富硒栽培生产。
Description
技术领域
本发明属于基因工程鉴定技术领域，具体涉及一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2及其应用。
背景技术
硒是动物和人类的必需微量营养元素，参与动物和人类多种酶的合成，缺硒会造成多种疾病。通过补硒可以增强人体免疫力，起到防癌抗癌、延缓衰老的作用。我国目前2/3的地区面临缺硒的问题，亟需通过硒生物强化的方式改变上述地区的现状。在自然界中，硒主要以无机和有机形式存在，其中有机硒更易被人体吸收且更加安全。植物作为饮食中硒的主要来源之一，其硒代谢对人体和动物营养至关重要。此外，研究发现，动物饲料中添加0.14mg/kg的硒可将人体血浆的硒含量提高约30μg/L，证明富硒饲料可作为人体补硒的间接方案之一。
由于硒与硫的化学性质相似，植物通过硫同化通路将外源硒转化为硒代氨基酸，其中包括蛋氨酸(Methionine,Met)的类似物–硒代蛋氨酸(Selenomethionine,SeMet)。植物中蛋氨酸合成可通过以下两个途径：其一，半胱氨酸在胱硫醚γ合酶(Cystathioninegamma-synthase,CGS)、胱硫醚β裂解酶(Cystathionine beta-lyase,CBL)和蛋氨酸合酶(Methionine synthase,MTR)的催化下可合成蛋氨酸。此外，植物可利用同型半胱氨酸(Homocysteine,HCys)作为底物，在同型半胱氨硫甲基转移酶(Homocysteine S-Methyltransferase,HMT)的催化下，以硫腺苷蛋氨酸(S-adenosylmethionine,S-adenosyl-Met)或硫甲基蛋氨酸(S-methylmethionine,S-MeMet)做为甲基供体合成蛋氨酸。
构树具有一定的硒积累能力，经外源硒处理的构树叶片中，硒代蛋氨酸(SeMet)为主要有机硒形态，具有较高的饲用价值。且硒代蛋氨酸合成的关键基因-同型半胱氨酸硫甲基转移酶基因(BpHMT2)的表达水平显著上调表达，因此解析BpHMT2基因的功能对提高构树中的有机硒含量、硒耐受能力及饲用价值具有重要意义。
发明内容
本发明的目的是针对现有的问题，提供了一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2及其应用。构树BpHMT2基因能够提高植物耐硒、硒代蛋氨酸和硒积累能力。
本发明是通过以下技术方案实现的：
一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2，其核苷酸序列如SEQ ID NO.1所示。
SEQ ID NO.1：
ATGGGAATCGGAAAAGCGACGTCGTTGGAGGAAGTGATAGAGAAGGCCGGAGGGTGCGCGGTGGTCGACGGCGGCTTCGCGACGCAGCTTGAGAGCCACGGCGCCGACATCAATGACCCTCTCTGGAGCGCTCTCTGCCTCATCAAAGACCCTCACCTCATCAAACTGGTCCACATGGAGTACTTGGAAGCCGGAGCTGACGTTTTGGTTAGCTCATCTTACCAGGCCACCATTCCAGGATTTCTGTCACGAGGACTATCAATTGAAGAAGCGGAATCTCTGCTGAAGAAGAGCGTGACGTTGGCGGTAGAAGCTCGCGATAGCTTCTGGGATGCTGCGAAGGCGAGCCCCGCGCATGGCTATAACAGGGCTCTGGTTGCAGCATCCATTGGAAGCTATGGAGCTTATCTTGCTGATGGCTCAGAATACAGCGGACGTTATGGACCAGACGTTGGTTTGGACAAGCTGAAGGATTTCCACAGACGAAGATTGCAGGTTCTCGTGGACGCCGGCGCGGATTTACTCGCGTTCGAGACCATTCCGAATAAGCTAGAAGCACAGGCTTGTATGGAGCTGCTTGAAGAAGAAAAAGTGCAAATACCATCTTGGATTTGCTTTAGCTCTGTAGATGGAGAGAAAGCCCCTTCAGGAGAGAGTTTCAAGGAGTGCCTTGACATTATAAACAAAAGTGACAAAGTTCATGCAGTGGGAATTAACTGTGCACCTCCTCATTTCATTGAGGATCTCATCAGAAAATTCAGTGAGCTTACAGAAAAAGCCATAGTTGTTTACCCAAATAGTGGTGAAGTCTGGGATGGCAGGGCCAAGAGATGGCTGCCATCAAAGTGTTCCGACGACAACAAATTTGAGTGTTTCGCAACAAGATGGCGAAATTCAGGAGCTAAACTGATTGGAGGGTGCTGTCGGACGACACCTTCCACCATTCAAGCTATTTCAAAAGCTTTGAAAGAATGGCCTTGA
进一步地，其其编码的蛋白的氨基酸序列如SEQ ID NO.2所示。
SEQ ID NO.2：
MGIGKATSLEEVIEKAGGCAVVDGGFATQLESHGADINDPLWSALCLIKDPHLIKLVHMEYLEAGADVLVSSSYQATIPGFLSRGLSIEEAESLLKKSVTLAVEARDSFWDAAKASPAHGYNRALVAASIGSYGAYLADGSEYSGRYGPDVGLDKLKDFHRRRLQVLVDAGADLLAFETIPNKLEAQACMELLEEEKVQIPSWICFSSVDGEKAPSGESFKECLDIINKSDKVHAVGINCAPPHFIEDLIRKFSELTEKAIVVYPNSGEVWDGRAKRWLPSKCSDDNKFECFATRWRNSGAKLIGGCCRTTPSTIQAISKALKEWP
一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2的应用，所述BpHMT2基因或蛋白在提高植物耐硒能力中的应用。
一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2的应用，所述BpHMT2基因或蛋白在提高植物硒积累能力中的应用。
一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2的应用，所述BpHMT2基因或蛋白在提高植物硒代蛋氨酸合成中的应用。
进一步地，所述植物包括拟南芥。
一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2的应用，所述BpHMT2基因或蛋白在培育富硒植物中的应用。
本发明相比现有技术具有以下优点：
本发明提供了一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2及其应用，构树BpHMT2基因能够提高植物的耐硒和聚硒能力；BpHMT2与构树硒代蛋氨酸合成相关，它能提高构树总硒和硒代蛋氨酸含量，以构树BpHMT2为提高构树聚硒能力的靶基因，可通过遗传育种方式，培育富硒构树来作为畜禽饲料原料，进而满足人们日常饮食中补硒的需求。
附图说明
图1为本发明提供的BpHMT2阳性克隆PCR检测结果图；
图2为本发明提供的BpHMT2基因编码区序列及预测的氨基酸序列图；
图3为本发明提供的pNC-Cam1304-MCS-35S::BpHMT2重组质粒图谱图；
图4为本发明提供的拟南芥纯合子筛选图；
图5为本发明提供的转基因拟南芥PCR检测图；
图6为本发明提供的拟南芥GUS染色显微观察照片；
图7为本发明提供的不同拟南芥株系在含硒酸钠平板培养后的生长情况；
图8为本发明提供的不同拟南芥株系叶喷硒酸钠处理后的生长情况；
图9为本发明提供的硒处理拟南芥的总硒、总硫、硒代蛋氨酸和Se(VI)含量图；
图10为本发明提供的不同拟南芥株系中BpHMT2相对表达水平图；
图11为本发明提供的不同拟南芥株系中AtAPS表达水平图；
图12为本发明提供的不同拟南芥株系中AtAPR表达水平图；
图13为本发明提供的拟南芥AtMMT和AtNiFS基因表达水平图；
图14为本发明提供的拟南芥AtSAM基因表达水平。具体实施方式
本发明提供了构树BpHMT2基因在提高植物硒耐受能力中的应用。转BpHMT2基因的拟南芥根系显著长于野生型和突变体。
本发明还提供了构树BpHMT2基因在提高植物总硒含量中的应用。转BpHMT2基因的拟南芥总硒含量均高于野生型和突变体。
本发明还提供了BpHMT2提高植物有机硒含量的应用。转BpHMT2基因的拟南芥硒代蛋氨酸含量均高于突变体和野生型。
在本发明中，所述植物包括拟南芥。
本发明还提供了构树BpHMT2基因在培育富硒植物中的应用。
下面结合具体实施例对本发明所述的构树BpHMT2基因的应用做进一步详细的介绍。
实施例1
材料：供试材料分别为哥伦比亚野生型(Columbia,Col-0)和hmt基因突变体(SALK_204527C)拟南芥，其中突变体购买自AraShare科技服务中心，野生型拟南芥为本申请的申请人所在实验室保存。
种子消毒：种子经75％酒精消毒30s，无菌水清洗2次，3％次氯酸钠消毒6min，无菌水清洗5～6次，经过消毒的拟南芥种子转至无菌滤纸上，种子干燥后转至含有MS的平板中，经4℃春化2d。随后转至光照培养室培养，生长2周后，移栽到育苗基质中(泥炭:草炭:珍珠岩＝7:2:1)，移栽后表面覆盖保鲜膜生长1周，当移栽苗抽薹至5-6cm后，剪去主茎促发更多侧茎，用于后续侵染。
利用根癌农杆菌GV3101介导遗传转化BpHMT2基因至hmt突变体拟南芥，分析转基因拟南芥的耐硒能力。
1.BpHMT2基因克隆及测序
(1)引物设计：
根据硒处理构树的转录组数据，利用HMT的隐马尔可夫模型(PF02574)比对构树中的HMT蛋白，并根据本申请人在前期实验中获得的构树硒含量和HMT基因的表达水平数据，分析相关性最高的BpHMT2基因，根据CDS序列设计引物BpHMT2-F和BpHMT2-R(表1)。
(2)总RNA提取：
选取构树叶片组织，使用使用RNA提取试剂盒(TaKaRa MiniBEST Plant RNAExtraction Kit,宝生物,中国)按照操作说明进行总RNA的提取，使用超微粒核酸浓度就检测仪(Nanodrop One spectrophotometer,赛默飞,美国)进行RNA浓度和质量的评估，其中选择OD260/OD280在1.8～2.1之间，RNA浓度不低于100ng/μL作为合格的样品。
(3)PCR扩增：
使用III 1st Strand cDNA Synthesis Kit(诺唯赞,南京)进行cDNA的合成，并以此为模版，利用2×Rapid Taq Master Mix预混酶进行PCR扩增。
(4)胶回收及测序：
电泳检测后使用胶回收试剂盒回收产物，使用TA克隆试剂盒将BpHMT2基因连接到pMD19-T载体，转化至DH5α大肠杆菌感受态，活化后摇菌1h，涂布于含有氨苄青霉素(Amp)抗性的LB固体培养基，挑取单菌落以M13-47和M13-48(表1)引物进行PCR验证，将阳性克隆(图1)进行测序。
测序后发现该片段长度981bp，编码326个氨基酸，分子量35.41KDa，其中Tyr74,Thr179为HMT酶活性的必需氨基酸。此外，Cys240,Cys307和Cys308共同构成了用于催化作用的Zn2+-binding中心，表明该蛋白具有HMT酶的完整结构，因此将该基因命名为BpHMT2。
表1本发明中所使用的引物序列
2.植物过表达载体pNC-Cam1304-MCS-35S::BpHMT2的构建
设计一步克隆引物BpHMT2-OE-F和BpHMT2-OE-R：(表1)，以测序比对成功的BpHMT2阳性克隆为模版，扩增含有部分载体同源臂的线性化基因。以SfiI内切酶酶切pNC-Cam1304-MCS-35S质粒，琼脂糖凝胶电泳检测酶切效果。随后使用一步克隆试剂盒(CloneExpression Ultra One Step Cloning Kit)连接线性化的基因及载体，转至DH5α大肠杆菌感受态，涂布于含有卡那霉素(Kan)的平板，使用M13-47和M13-48检测阳性克隆()。将阳性菌活化后按照质粒提取试剂盒(EasyPure Plasmid MiniPrep Kit,全式金,北京)说明书提取pNC-Cam1304-MCS-35S::BpHMT2质粒，用于后续的农杆菌转化。
3.pNC-Cam1304-MCS-35S::BpHMT2质粒转入农杆菌
取2.5μL从步骤2提取的质粒，加入至50μL GV3101根癌农杆菌感受态中，吸打混匀后转至预冷的电击杯，2500V电击转化，于超净工作台加入800μLYEP液体培养基，28℃，200rpm复苏2h。取30μL菌液涂布于含有卡那霉素(50mg/L)的YEP平板，28℃倒置培养2～3d。挑取单菌落，用M13-47和M13-48引物鉴定阳性克隆，获得转pNC-Cam1304-MCS-35S::BpHMT2的GV3101根癌农杆菌。
4.花序侵染法转拟南芥
4.1侵染液准备
吸取活化的含有pNC-Cam1304-MCS-35S::BpHMT2质粒的GV3101农杆菌，接种至含有25mg/L利福平(Rif)和50mg/L Kan的YEP液体培养基，28℃，200rpm，过夜培养。取500μL过夜培养的菌液于30mL含Rif和Kan的YEP培养基至OD600值为0.6～0.8。5000rpm，室温离心15min，弃上清，使用质量分数5％的蔗糖溶液重悬菌体，调节OD600值在0.8～1.0左右。
4.2花序侵染
侵染hmt突变体拟南芥之前剪去花梗上已伸出的柱头和果荚，将拟南芥花序完全浸泡在侵染液中30s。转化后，用保鲜膜罩住转化植株，保持湿润，于室温下黑暗培养16h，随后对植株喷水冲洗表面活性剂及残留菌体，转至光照培养箱，设定光周期14h/10h，温度25℃/22℃，湿度70％。7d后重复侵染一次，以提高转化率，随后正常培养至种子成熟。
5.转BpHMT2基因拟南芥纯合子筛选和鉴定
5.1转基因拟南芥纯合子筛选
按照实施例1的拟南芥消毒方法进行拟南芥种子的消毒，随后均匀的播撒于含有30mg/L潮霉素(Hyg)的MS平板中，封口膜密封，将平板置于4℃冰箱春化2d后转至光照培养室培养，设定光周期14h/10h，温度25℃，光照强度2000Lux，拟南芥幼苗生长12d左右(图4)，将根系正常生长和出现真叶的拟南芥(T1代)移栽至培养基质中，置于光照培养箱中培养，培养条件与步骤4.2中一致。
5.2拟南芥突变体纯合体PCR鉴定
取光照培养箱生长一个月左右的转基因拟南芥叶片，使用CTAB法获得粗提的DNA。并以DNA为模版使用BpHMT2-OE-F和BpHMT2-OE-R引物()检测转基因拟南芥是否为阳性植株(图5)。将转基因拟南芥继续培养并重复5.1的操作至收获T3代纯合型转BpHMT2基因的拟南芥。
5.3GUS染色鉴定
分别取生长于MS培养基中约两周的野生型和转基因拟南芥幼苗，置于1.5mL离心管中，按照Gusblue kit(华越洋，北京)试剂盒说明书加入预混的GUS染液淹没叶片，于37℃恒温培养箱中孵育12h。随后倒出染液，并用75％酒精脱色，每8h更换一次酒精，直至叶片呈白色透明状。使用体式显微镜(Leica M165 FC，莱卡)观察染色情况并拍照记录。
实施例2
利用外源施硒酸钠的方式处理拟南芥，通过比较野生型、突变体和转基因株系的生长情况、总硫、总硒含量及硒形态数据分析构树BpHMT2基因对植物硒积累能力的影响。
1.含硒酸钠平板培养拟南芥
对Col-0、hmt和3个BpHMT2过表达拟南芥株系OE01-OE03种子(T3代)进行消毒后，播种于含有400μmol/L硒酸钠的MS培养基，4℃春化24h后竖立放置，以使拟南芥根系沿培养基表面生长。培养条件与步骤4.2一致。播种15d以后测量不同拟南芥植株的根长情况并拍照记录。结果显示，Col-0野生型拟南芥的平均根长为1.34cm，hmt突变体的根长为1.33cm，3个过表达株系(OE01-OE03)的根长分别达到4.96、4.14和4.08cm()，显著高于野生型和突变体(图7)，表明BpHMT2基因能够促进拟南芥根系的伸长，进而提高突变体拟南芥对硒酸钠的耐受能力。
表2含硒酸钠平板中生长的不同拟南芥株系根长情况
2.拟南芥叶喷硒酸钠
采用实施例1中的方法对野生型，突变体及3个过表达株系拟南芥的T3代种子进行消毒，播种于MS培养基，出现4片真叶后移栽至基质中培养。培养20d左右，选择生长一致的拟南芥，每个处理设置3个重复，每个重复30株。用400μmol/L的硒酸钠溶液喷施拟南芥叶片，喷适量以刚好有液滴滴下为止，每5d处理一次，共处理3次。采收前2d用去离子水冲洗叶片正反面，去掉残留的硒。液氮速冻拟南芥叶片后于-80℃保存，用于后续生理和定量试验。
3.总硫和总硒含量测定
3.1总硫含量
使用微机库仑测硫仪(CLS-3000D,江苏国创,中国)通过电解法测定，称取碘化钾和溴化钾各5.0g，加入200mL水溶解后添加10mL的冰乙酸并定容至250mL。精确称取0.2g干燥的构树叶片粉末，加入到77mm×6mm的瓷舟中，设置裂解炉温度900℃，气流量1.0L/min，阶段1检测45s，阶段2检测30s，阶段三检测225s。结果采用峰面积的积分表示，每个样品重复检测3次。结果显示，Col-0和hmt株系的总硫含量分别为0.83和0.87mg/kg DW，株系OE01-OE03的总硫含量分别为1.40、1.49和2.10mg/kg DW(图9b)，均显著高于野生型和突变体株系，表明构树BpHMT2基因显著提高拟南芥的硫积累。
3.2总硒含量
总硒含量的测定采用HG-AFS进行，称取0.2g干燥的构树粉末，添加到消解管中，添加10mL硝酸和2mL过氧化氢，加盖密封后，于微波消解仪中梯度消解55min，消解结束冷却后，向管中添加5mL HCl(6mol/L)继续加热至溶液变为清亮无色并伴有白烟出现，冷却后转移至10mL容量瓶中，加入2.5mL铁氰化钾溶液(100g/L)，随后用水补足到10mL，待测。吸取硒标准液分别配制0、5、10、20、30μg/L的硒标准系列溶液，用以绘制标准曲线。硒灯总电流60mA，负高压280V。结果显示，Col-0和hmt拟南芥的总硒含量分别为65.42和90.11μg/gDW，两者无显著差异；转基因株系OE01-OE03的总硒含量分别达到166.04、190.86和130.53μg/gDW(图9a)，相比hmt突变体分别提高了0.84、1.12和0.45倍，显著高于Col-0和hmt株系，表明构树BpHMT2基因能够提高hmt突变体拟南芥的硒积累能力。
4.硒形态测定
硒形态的检测采用LC-AFS进行，用0.01mol/L的磷酸缓冲液配制8mg/mL的蛋白酶XIV，称取0.2g构树干粉，添加8mL蛋白酶XIV至10mL离心管中，37℃振荡孵育16h，随后室温超声提取30min，于10000rpm离心10min(4℃)。上清经0.45μm水系滤膜过滤后用于检测。分别配制AB流动相，其中A流动相为5mM柠檬酸(pH 4.6)，B流动相为40mM的磷酸二氢钾(pH6.0)。梯度程序：A相8min，A->B相切换1min，B相6min，B->A相1min。分别配制SeCys2、SeMeCys、Se(IV)、SeMet、Se(VI)标准溶液，并稀释为40、80、120、160、200μg/L(以Se计)的梯度标准溶液。采用Hamilton PRP-X(汉密尔顿,美国)阴极色谱柱，柱温25℃，氩气(99.999％)作为载气，流速600mL/min，负高压320V，总灯电流80mA。每组样品设计3次生物学重复，每个样品测定3次。硒酸钠处理的拟南芥中共检测到两种硒形态，分别为SeMet和Se(VI)。Col-0和hmt株系的SeMet含量分别为14.26和16.41μg/g DW，转基因株系分别达到23.44、30.70和31.62μg/g DW，相比hmt株系分别提高了0.43、0.87和0.93倍(图9c)；此外，hmt株系的Se(VI)含量高于Col-0株系，达到107.15μg/g DW，3个过表达株系的六价硒含量分别为160.52、158.39和207.15μg/g DW，均显著高于野生型，且OE03的六价硒含量显著高于hmt株系(图9d)。综上所述，BpHMT2能够显著提高拟南芥对硒的吸收能力，提高拟南芥中SeMet和Se(VI)的含量，表明BpHMT2具有催化SeMet合成的功能。
实施例3
对硒处理拟南芥中的硒代谢相关基因进行实时定量荧光PCR检测，分析构树BpHMT2基因对植物硒代蛋氨酸合成通路基因的影响。
1.拟南芥RNA提取及反转录
使用实施例1中步骤1的方法提取实施例2中硒酸钠叶喷处理的Col-0，hmt和3个转基因拟南芥株系的叶片RNA并反转录获得cDNA。
2.拟南芥硒代谢基因实时定量PCR检测
从EnsemblPlants(https://plants.ensembl.org/Arabidopsis_thaliana/Info/Index)网站下载拟南芥TAIR10基因组数据，依据植物硒代谢通路，从拟南芥基因组中选取硒代谢通路中的三磷酸腺苷硫酸化酶(AtAPS)、三磷酸腺苷硫酸盐还原酶(AtAPR)、S-腺苷蛋氨酸合成酶(SAM)、半胱氨酸裂解酶(AtNiFS)和蛋氨酸甲基转移酶(AtMMT)基因，使用Tbtools提取相应基因的CDS序列，并使用Primer Premier 6.0设计包括BpHMT2基因的定量检测引物()。以步骤1反转录的各拟南芥株系cDNA为模版，以PP2A为内参基因，进行qRT-PCR检测。
结果显示，Col-0和hmt中未检测到BpHMT2的表达，3个过表达株系均检测到BpHMT2的表达，且OE03中的表达水平显著高于OE01和OE02(图10)，与硒形态结果中的SeMet和Se(VI)含量结果一致。此外，AtAPS1和AtAPS3的表达水平在hmt和转基因株系中均显著低于Col-0株系。AtAPS2和AtAPS4的表达水平则在Col-0和hmt中无显著差异，且在转基因株系中的表达水平均低于hmt株系，其中AtAPS2的表达水平显著低于hmt株系(图11)。AtAPR的定量结果显示，3个AtAPR表达水平在hmt株系和转基因株系中均显著低于Col-0株系，且3个AtAPR基因的表达水平在hmt株系和转基因株系中无显著差异(图12)。AtNiFS和AtMMT1的表达情况相似，其中hmt株系中的AtMMT1表达水平相比野生型降低了约24倍，且各转基因株系与hmt株系间无显著差异(图13)；而AtNiFS的表达水平在hmt株系和转基因株系均显著低于Col-0株系，且转基因株系的AtNiFS表达水平略高于hmt株系(图13)。AtSAM基因的表达水平在hmt株系和各转基因株系中均显著低于Col-0株系，且AtSAM1、AtSAM2和AtSAM3在hmt株系和转基因株系间无显著差异，但AtSAM4的表达水平在所有转基因株系中均显著高于hmt株系(图14)。在植物中，SAM催化SeMet形成腺苷硒代蛋氨酸(Adenoyl-SeMet)，其进一步作为HCys的甲基供体，在HMT的催化下形成SeMet，因此，BpHMT2主要利用AtSAM4催化合成的Adenoyl-SeMet作为甲基供体，从而提高转基因拟南芥中的SeMet含量。
综上所述，BpHMT2提高了hmt突变体拟南芥的耐硒能力，并且提高拟南芥的硒积累能力，并提高了有机硒成分硒代蛋氨酸的含量。说明BpHMT2是参与硒代蛋氨酸合成的关键基因，并且能提高植物对硒的积累和耐受能力。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，都应涵盖在本发明的保护范围之内。
Claims (6)
1. 一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2，其特征在于，其核苷酸序列如SEQ ID NO.1所示。
2. 根据权利要求1所述一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2，其特征在于，其其编码的蛋白的氨基酸序列如SEQ ID NO.2所示。
3. 一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2的应用，其特征在于，所述BpHMT2基因或蛋白在提高植物耐硒能力中的应用，所述BpHMT2基因的序列如SEQ ID NO.1所示，所述BpHMT2蛋白的序列如SEQ ID NO.2所示，所述植物为构树或拟南芥。
4. 一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2的应用，其特征在于，所述BpHMT2基因或蛋白在提高植物硒积累能力中的应用，所述BpHMT2基因的序列如SEQ ID NO.1所示，所述BpHMT2蛋白的序列如SEQ ID NO.2所示，所述植物为构树或拟南芥。
5. 一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2的应用，其特征在于，所述BpHMT2基因或蛋白在提高植物硒代蛋氨酸合成中的应用，所述BpHMT2基因的序列如SEQ ID NO.1所示，所述BpHMT2蛋白的序列如SEQ ID NO.2所示，所述植物为构树或拟南芥。
6. 一种构树同型半胱氨酸硫甲基转移酶基因BpHMT2的应用，其特征在于，所述BpHMT2基因或蛋白在培育富硒植物中的应用，所述BpHMT2基因的序列如SEQ ID NO.1所示，所述BpHMT2蛋白的序列如SEQ ID NO.2所示，所述植物为构树或拟南芥。
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