CN116926108A - 向日葵尿黑酸植基转移酶基因HaVTE2在提高植物耐盐性能中的应用 - Google Patents
向日葵尿黑酸植基转移酶基因HaVTE2在提高植物耐盐性能中的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及向日葵尿黑酸植基转移酶基因HaVTE2在提高植物耐盐性能中的应用,或在培育具有耐盐性能的转基因植物中的应用;所述向日葵尿黑酸转移酶基因HaVTE2,其编码区的核苷酸序列如SEQ ID NO.1所示。本发明通过在植物细胞或种子中过量表达向日葵尿黑酸植基转移酶基因HaVTE2,可以获得耐盐性能优于野生型植株的转基因植物,这有利于在盐胁迫下提高植物的出苗率和成苗率,以及缩短出苗时间,提高存活率,长得更好。本发明为改造或培育其他耐盐的农作物新品种提供了理论依据和基因来源,有助于从根源上提高农作物的耐盐性能。
Description
技术领域
本发明属于生物技术领域,具体涉及向日葵尿黑酸植基转移酶基因HaVTE2在提高植物耐盐性能中的应用。
背景技术
土壤盐渍化是指易溶性盐分在土壤表层逐渐累积,最终以晶体形式析出,是非常严峻的非生物胁迫之一,是农业生产中影响作物产量的主要限制因素。盐胁迫主要通过渗透胁迫、离子毒害、亚细胞水平的氧化胁迫及次生胁迫对植物体内细胞膜完整性和各种酶活性产生重大影响,阻碍植物对水分和养料的吸收与利用,造成膜功能紊乱和细胞死亡,最终导致植物在盐胁迫下的种子萌发、叶片和根的生长、株高和果实发育等均受到显著抑制。
种子萌发是从物理性的吸水阶段开始,进行一系列物质的转化与合成,经过一段时间,胚根突破种皮并继续伸长,表明种子已开始萌发。种子萌发期是作物生长发育的第一阶段,在盐渍环境下,种子萌发也会最先受到盐胁迫的影响。种子萌发在盐胁迫下,始终会受到渗透胁迫和离子毒害作用的影响,具体表现为,种子萌发速率和发芽率随盐胁迫浓度的增加而降低。种子萌发期受盐胁迫会直接影响种子发芽出苗,呼吸作用受到损害,光合作用被抑制,导致植株生长受到严重阻碍,作物产量、质量均下降。因此,通过基因工程改良和提高植物耐盐能力具有重要意义与应用价值。
发明内容
本发明的目的在于提供向日葵尿黑酸植基转移酶基因HaVTE2在提高植物耐盐性能中的应用。
本发明是通过以下技术方案实现的:
向日葵尿黑酸转移酶基因HaVTE2,其编码区的核苷酸序列如SEQ ID NO.1所示,所述SEQ ID NO.1由1194个核苷酸组成。
SEQ ID NO.1:
ATGAAGTCTTTGATTCTTGGGTCTTTTTCTTCGTACAAGGTTTCTACTTATTC
TCTGCCGTCACCAGTTTCATCTTCTTCACTTGTATCATCAGGTTGTTATAATG
TATCATCACTAGGAGCATCAAAGAATAAAGGAATCGTCCAATCTCAATCAA
GTTTTTTGAGATGCAATACGGACAGAACTAATAAAAGTTTTCTACTTTCTCA
CAAATTCAACACACAACGCGTTGCAAGTGCGATTTCTGAACAACCTGTTGA
TCCTGATCCCACAAGTCCTCAACAATCATTACCAAATGCTATAAATGCTTTC
TATAGGTTTTCAAGACCTCACACAGTTATAGGAACTGCATTGAGCATAGTTT
CAGTTTCACTCCTTGCAGTTCAAAAGCTTTCAGACTTTTCTCCATCATTCTT
CATTGGTGTTTTGGAGGCAATTGTTGCTGCCTTCTTTATGAATATATATATTGT
TGGATTGAACCAGTTATCTGATATAGAAATAGACAAGGTTAACAAGCCCTAT
CTTCCGTTGGCATCTGGAGAATATTCCGTTAAAACTGGGATTATTATTGTATC
CTCATTTGCATTCATGAGTTTCTGGCTTGGATGGATTGTTGGTTCATGGCCTT
TATTTTGGGCACTCTTCATAAGTTTTCTTCTTGGGACGGCGTATTCAATCAAT
ATGCCGATGCTGAGATGGAAGCGATTTGCTCTCGTGGCAGCAATGTGCATT
CTAGCTGTAAGAGCTGTAATAGTTCAAATCGCATTTTACCTACACATTCAGA
CTTTTGTGTACGGAAGACTCGCTGTGTTCCCAAAACCCGTGATATTTGCAA
CCGGATTTATGAGTTTCTTCTCTGTTGTTATAGCATTGTTTAAGGACATACCT
GACATTGTTGGAGACAAGATCTTCGGCATTCAATCATTTACCGTCCGCTTGG
GTCAAAAGCGGGTGTTTTGGATCTGTATTTTATTACTTGAAGTGGCTTATGC
TGTTGCTATTCTAGTTGGGGCATCATCTCCCTTCCTTTGGAGCAGATACATA
ACGGTATTGGGTCACGCGATTCTTGGTCTAATACTTTGGGGTCGCGCGAAAT
CAATCGATTTGGAGAACAAATCAGCTATAACCTCATTTTACATGTTCATATG
GCAGTTGTTCTACGCCGAGTACTTGCTCATACCGCTTGTGAGGTGA
向日葵尿黑酸植基转移酶基因HaVTE2,其编码的蛋白质的氨基酸序列如SEQ IDNO.2所示,所述的SEQ ID NO.2由397个氨基酸残基组成。
SEQ ID NO.2:
MKSLILGSFSSYKVSTYSLPSPVSSSSLVSSGCYNVSSLGASKNKGIVQSQSSFL
RCNTDRTNKSFLLSHKFNTQRVASAISEQPVDPDPTSPQQSLPNAINAFYRFSR
PHTVIGTALSIVSVSLLAVQKLSDFSPSFFIGVLEAIVAAFFMNIYIVGLNQLSDI
EIDKVNKPYLPLASGEYSVKTGIIIVSSFAFMSFWLGWIVGSWPLFWALFISFLL
GTAYSINMPMLRWKRFALVAAMCILAVRAVIVQIAFYLHIQTFVYGRLAVFPKP
VIFATGFMSFFSVVIALFKDIPDIVGDKIFGIQSFTVRLGQKRVFWICILLLEVAY
AVAILVGASSPFLWSRYITVLGHAILGLILWGRAKSIDLENKSAITSFYMFIWQL
FYAEYLLIPLVR
向日葵尿黑酸植基转移酶基因HaVTE2在提高植物耐盐性能中的应用,或在培育具有耐盐性能的转基因植物中的应用。本发明通过研究发现,向日葵尿黑酸植基转移酶基因HaVTE2能够显著提高转基因植物在萌发期的耐盐性能。本发明为培育农作物新品种提供了理论依据和基因来源,有助于从根源上提高植物的耐盐性能。
上述应用,在具体应用时,将含有向日葵尿黑酸植基转移酶基因HaVTE2的表达载体转入植物细胞或种子,使向日葵尿黑酸植基转移酶基因HaVTE2过表达,从而获得稳定可遗传的耐盐转基因植株。
所述过表达基因序列为:向日葵尿黑酸植基转移酶基因HaVTE2基因的CDS全长序列,共1194bp。
进一步地,还包括收集所述稳定可遗传的耐盐转基因植株的种子,再用所述种子繁育获得后代种子,通过测序验证获得纯合的过表达的后代种子长成的植株。
所述测序验证的方法是,提取上述纯合的过表达的后代种子长成的植株的基因组DNA,并用688-seq-F和CD3-OCS-seqR引物进行PCR扩增并测序:
688-seq-F:GGGATGACGCACAATCCCAC,
CD3-OCS-seqR:GAATGAACCGAAACCGGCGG。
一种植物表达载体,其含有上述向日葵尿黑酸植基转移酶基因HaVTE2。
一种遗传工程化的宿主细胞,其含有上述植物表达载体,或其基因组中插入了向日葵尿黑酸植基转移酶基因HaVTE2。
所述遗传工程化的宿主细胞的构建方法,是将上述植物表达载体导入宿主细胞中,使植物表达载体/向日葵尿黑酸植基转移酶基因HaVTE2在宿主细胞中有效表达。
上述植物表达载体、遗传工程化宿主细胞在培育具有耐盐性能的转基因植物中的应用。
上述提及的植物为双子叶植物。优选地,所述双子叶植物为拟南芥、向日葵、白菜或甘蓝。
综上所述,本发明具有以下有益效果:
1.本发明通过将向日葵尿黑酸植基转移酶基因HaVTE2连接于表达载体上,并利用农杆菌侵染法转化拟南芥,结果表明,转基因拟南芥在盐胁迫下的萌发率显著高于野生型拟南芥,说明向日葵尿黑酸植基转移酶基因HaVTE2能够显著提高转基因植物在萌发期的耐盐性能。
2.通过在植物细胞或种子中过量表达向日葵尿黑酸植基转移酶基因HaVTE2,可以获得耐盐性能优于野生型植株的转基因植物,这有利于在盐胁迫下提高植物的出苗率和成苗率,以及缩短出苗时间,提高存活率,长得更好。本发明为改造或培育其他耐盐的农作物新品种提供了理论依据和基因来源,有助于从根源上提高农作物的耐盐性能。
附图说明
图1为转基因拟南芥PCR鉴定电泳图。
图2为用测序引物688-seq-F的测序峰图。
图3为用测序引物CD3-OCS-seqR的测序峰图。
图4为不同测序引物的测序示意图。
图5为参考序列与测序序列比对图;图中sbjct表示NCBI网站上的参考序列,Query-表示转基因拟南芥的测序序列,A图中Query表示用测序引物688-seq-F所测得的序列,B图中Query表示用测序引物CD3-OCS-seqR所测得的序列。
图6为转基因拟南芥RNA鉴定。
图7为转基因拟南芥Western Blot电泳图。
图8为转基因拟南芥的耐盐表型;图中Col表示野生型拟南芥,1/5/6/8/11为转基因拟南芥。
图9为200mM NaCl处理下的萌发统计图;图中Col为野生型拟南芥,#1/5/6/8/11为转基因拟南芥;P<0.05,具有差异,凡有一个相同标记字母的即为差异不显著,凡具不同标记字母的即为差异显著。
具体实施方式
下面以具体实施例对本发明的技术方案做进一步说明,但是实施例具体细节仅为了说明本发明,并不代表本发明构思下全部技术方法。因此不应理解为对本发明总的技术方案限定。
下述实施例中的实验方法,如无特殊说明,均为常规方法,所用的试验材料,如无特殊说明,均为常规生化试剂与药品,实验均设置三次重复以上,结果取平均值。
表1实施例中采用的引物名称及序列
实施例1:过表达HaVTE2基因的转基因拟南芥的构建
一、HaVTE2表达载体(688-HaVTE2)的构建
1.目的基因的获得:
以向日葵cDNA为模板、引物HaVTE2-CDS-F和HaVTE2-CDS-R进行PCR扩增,胶回收纯化,从而获得HaVTE2基因片段。PCR反应体系如表2。
表2PCR反应体系(50μL):
PCR程序设定为:95℃预变性3min;95℃变性15s,56℃退火15s,72℃延伸60s,35个循环;72℃后延伸5min。
2.用BamHI酶切688表达载体,并进行胶回收纯化;用重组法构建688-HaVTE2表达载体,配好重组体系后置于金属浴50℃,反应15min。重组体系如表3。
表3重组体系(5μL):
3.质粒转化:
将上述重组产物转入50μL大肠杆菌感受态细胞,轻弹混合后冰上静30min,42℃,1min,转冰上静置2min。加入500μL LB培养基,37℃,200rpm,孵育1h,涂在LB+Kana的固体培养基上,倒置于37℃恒温培养箱过夜培养。挑取单克隆菌液PCR,鉴定成功后用(Vazyme)试剂盒提质粒,用引物688-seq-F和CD3-OCS-seqR进行测序,结果如图5所示,HaVTE2基因测序序列与参考序列一致,表明688-HaVTE2表达载体构建成功,即获得表达载体688-HaVTE2;随后将表达载体转入农杆菌GV3101中,用于后续的遗传转化。
二、拟南芥的遗传转化
挑取已经转化了688-HaVTE2质粒的农杆菌单菌落,接种到3ml的含有Kan和rif抗生素的LB培养基中,28℃,过夜培养,第二天转100ml含有Kan和rif抗生素的LB培养基中扩大培养,直至菌液OD600=0.6-0.8,4000rpm,20min,收菌;将菌重悬于适当的渗透液(1/2MS+10%蔗糖+400μL Silwet-77,OD600=0.8-1.0)中。在侵染前一天,给待侵染拟南芥浇水;并将生长了5周的拟南芥果荚剪去,保留部分拟南芥花。将植株的地上部分浸入上述侵染菌液中,20s-30s,轻微震荡;暗培养1天,然后置于正常培养条件(光照16h、黑暗8h);培养到植物成熟、收种子;将种子置于37℃烘干,用75%乙醇和84消毒液消毒后,4℃春化3天,将种子点在带有Basta抗性的MS培养基上筛选阳性苗。
三、转基因拟南芥的鉴定
通过CTAB法提取植株叶片的基因组DNA,以688-seq-F和CD3-OCS-seqR引物进行PCR扩增,扩增程序如下:94℃预变性4分钟,94℃变性40秒,58℃退火30秒,72℃延伸1分钟,扩增30个循环,最后再72℃延伸5分钟。实施例2:过表达HaVTE2基因的转基因拟南芥萌发期的耐盐实验
本发明所用生长实验的流程为:以野生型拟南芥(COL)和转基因拟南芥为实验材料,随机挑选种子,37℃烘2-3天,接着用乙醇和84消毒液对野生型和转基因拟南芥种子进行消毒,随后用无菌水置于4℃春化3天,将春化好的种子分别点在MS培养基和添加200mMNaCl的MS培养基上,22℃,光照16h/黑暗8h,每个Line各36粒种子,重复3次,观察并在生长4-5天后拍照记录表型(图8)和统计萌发率(图9),其中野生型拟南芥平均萌发率44.44%,转基因拟南芥#1/5/6/8/11的萌发率分别为86.11%/77.78%/72.22%/83.33%/94.44%,转基因拟南芥的平均萌发率皆高于野生型拟南芥,且都具有显著差异。
实验表明,在200mM NaCl处理条件下,过表达HaVTE2基因的转基因拟南芥的萌发率明显高于野生型拟南芥,这有利于在盐胁迫下提高植物的出苗率和成苗率,以及缩短出苗时间,提高存活率,长得更好。
Claims (10)
1.向日葵尿黑酸植基转移酶基因HaVTE2在提高植物耐盐性能中的应用,或在培育具有耐盐性能的转基因植物中的应用;所述向日葵尿黑酸转移酶基因HaVTE2,其编码区的核苷酸序列如SEQ ID NO.1所示。
2.向日葵尿黑酸植基转移酶基因HaVTE2在提高植物耐盐性能中的应用,或在培育具有耐盐性能的转基因植物中的应用;所述向日葵尿黑酸植基转移酶基因HaVTE2,其编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述的应用,其特征在于,具体应用时,将含有向日葵尿黑酸植基转移酶基因HaVTE2的表达载体导入植物细胞或种子,使向日葵尿黑酸植基转移酶基因HaVTE2过表达,从而获得稳定可遗传的耐盐转基因植株。
4.根据权利要求3所述的应用,其特征在于,还包括收集所述稳定可遗传的耐盐转基因植株的种子,再用所述种子繁育获得后代种子,通过测序验证获得纯合的过表达的后代种子长成的植株。
5.植物表达载体,其含有向日葵尿黑酸植基转移酶基因HaVTE2,所述向日葵尿黑酸转移酶基因HaVTE2,其编码区的核苷酸序列如SEQ ID NO.1所示。
6.一种遗传工程化的宿主细胞,其含有权利要求5所述的植物表达载体,或其基因组中插入了向日葵尿黑酸植基转移酶基因HaVTE2。
7.权利要求5所述的植物表达载体在培育具有耐盐性能的转基因植物中的应用。
8.权利要求6所述的遗传工程化的宿主细胞在培育具有耐盐性能的转基因植物中的应用。
9.根据权利要求1~4任一项所述的应用,其特征在于,所述植物为双子叶植物。
10.根据权利要求9所述的应用,其特征在于,所述双子叶植物为拟南芥、向日葵、白菜或甘蓝。
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