CN115094082B - 鉴定MsPDS基因的VIGS沉默体系及鉴定方法 - Google Patents
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Abstract
本发明公开了鉴定MsPDS基因的VIGS沉默体系及鉴定方法。本发明所提供的鉴定MsPDS基因的VIGS沉默体系,包含:将SEQ ID NO.1所示的核苷酸序列或应用SEQ ID NO.2‑3所示的核苷酸序列扩增紫花苜蓿的cDNA得到的目的基因插入到VIGS沉默载体上得到的重组载体。利用本发明提供的沉默体系以及鉴定方法可以根据植株出现光漂白的频率,快速且高效的验证紫花苜蓿MsPDS基因功能,为紫花苜蓿遗传改良和分子育种相关的研究提供有力支持。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种紫花苜蓿MsPDS沉默体系及鉴定方法。
背景技术
紫花苜蓿(Medicago sativa L.)是一种多年生草本豆科植物,由于其高营养品质、产量和适应性,使得它是世界上最重要的饲料作物之一。由于紫花苜蓿遗传转化的复杂性,传统的基因功能鉴定方法大多都操作复杂,转化效率低,很难实现基因功能的快速高通量鉴定。病毒诱导的基因沉默(Virus-induced gene silencing,VIGS)技术是近20年发展起来的能够快速鉴定基因功能的反向遗传学重要方法之一,是植物体中天然存在的一种抵御外源核酸入侵的防御系统,正常情况下保护植物免受病毒的侵染。植物的这种防御机制可被病毒RNA激活,属于转录后基因沉默现象。如果在病毒载体中插入目标片段,侵染寄主植物后,植物会表现出目标基因功能丧失或表达水平下降的表型。利用这种机制,可以确定基因功能。这种技术是一种简单、快速、高通量的分析已知序列基因功能的方法。
研究紫花苜蓿抗病,抗旱,耐寒等抗逆基因对于紫花苜蓿的遗传改良,高效育种具备重要意义。目前在紫花苜蓿中使用的转基因方法鉴定基因功能耗时较长,耗费较大,缺乏一种快速,高效的鉴定体系。
发明内容
为了解决现有技术中存在的问题,实现简单、快速、高通量分析、鉴定紫花苜蓿MsPDS基因,本发明提供一种普适、快速且高效的验证紫花苜蓿相关基因功能的体系及其构建方法,为紫花苜蓿遗传改良和分子育种相关的研究提供基础。
为实现本发明的技术目的,本发明第一方面提供一种鉴定紫花苜蓿MsPDS基因的VIGS沉默体系,包括:
将SEQ ID NO.1所示的核苷酸序列插入到VIGS沉默载体上得到的重组载体。
其中,所述鉴定紫花苜蓿MsPDS基因的VIGS沉默体系还可以将SEQ ID NO.2-3所示的核苷酸序列扩增紫花苜蓿cDNA得到的目的基因插入到VIGS沉默载体上得到的重组载体得到。
其中,所述VIGS沉默载体为TRV2。
为实现本发明的技术目的,本发明第二方面提供一种构建上述鉴定MsPDS基因的VIGS沉默体系的方法,包括:
应用SEQ ID NO.2-3所示的碱基序列对紫花苜蓿的cDNA扩增,得到目的基因片段;
使用限制性内切酶EcoRI、BamHI同时对目的基因和VIGS沉默载体进行酶切,连接后转化大肠杆菌DH5α,并将筛选的阳性菌落转化到农杆菌GV3101,得到鉴定MsPDS基因的VIGS沉默体系。
其中,目的片段的长度为400-410bp,优选为404bp。
为实现本发明的技术目的,本发明第三方面提供鉴定MsPDS基因的方法,使用上述的鉴定MsPDS基因的VIGS沉默体系实现。
其中,上述方法包括:
将处于苗龄的紫花苜蓿植株置于鉴定MsPDS基因的VIGS沉默体系中,并在真空压强<0.1Mpa的条件下侵染;
侵染完成后,将其取出移栽至苗钵中使其继续生长,并观察叶片颜色变化。
特别是,所述苗龄为紫花苜蓿幼苗生长至第三~五片真叶。
优选的,所述苗龄为紫花苜蓿幼苗生长至第四片真叶。
特别是,所述侵染的真空压强为0.08Mpa。
特别是,所述侵染的时间为10-20min。
优选的,所述侵染的时间为20min。
特别是,所述待测紫花苜蓿植株为中苜1号或公农1号。
优选的,所述待测紫花苜蓿植株为中苜1号。
附图说明
图1是本发明实施例1中MsPDS的PCR扩增结果;
图2是本发明实施例1中MsPDS测序比对结果;
图3是本发明实施例1中农杆菌质粒pTRV2-MsPDS的PCR检测结果;
图4是本发明实施例1中中苜一号植株侵染后的表型照片;
图5是本发明试验例1中沉默后的植株表型,其中,A-C:‘中苜1号’紫花苜蓿沉默后的植株表型,A:对照;B和C:处理;D-F:‘公农1号’紫花苜蓿沉默后的植株表型,D:对照;E和F:处理。
具体实施方式
下面结合具体实例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明所讲授的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
本发明所用的试剂及仪器如无特别说明,均为市售产品,其中,本发明使用的pTRV-1和pTRV2质粒由丹麦奥胡斯大学提供。
实施例1
1、目的基因片段的获得及沉默载体构建
使用艾德莱试剂盒提取紫花苜蓿RNA,提取步骤按照试剂盒说明书进行,并按照艾德莱反转录试剂盒说明书将RNA反转录成cDNA。使用蒺藜苜蓿MtPDS基因的序列与“中苜1号”紫花苜蓿参考基因组进行BLAST分析,找到紫花苜蓿MsPDS基因(MsG0380016167.01),根据MsPDS的序列到NCBI中确定CDS区,如SEQ ID NO 1所示,并设计特异性引物,设计引物时引入EcoRI、BamHI酶切位点,添加后的引物如下:
序列编号 | 引物名称 | 核苷酸序列 |
SEQ ID NO.2 | PDS-Fl | GAATTCTCGATTTGATTTTCCCGAAG |
SEQ ID NO.3 | PDS-Rl | GGATCCCACCGCCCAAGGACTTAATA |
以紫花苜蓿的cDNA为模板,PDS-F1/R1为引物扩增出404bp大小的目的片段,如图1所示,PCR扩增产物用琼脂糖凝胶回收试剂盒(艾德莱)回收,得到PDS扩增产物。
采用限制性内切酶EcoRI、BamHI同时对PDS扩增产物和pTRV2进行双酶切,反应体系为:总体积50μL,Cut smart Buffer 5μL,质粒pTRV2 15μL,EcoR I 1μL,BamHI 1μL,ddH2O 28μL;Cut smart Buffer 5μL,PCR扩增产物25μL,EcoRI 1μL,BamHI 1μL,ddH2O 18μL;37℃反应30min后进行胶回收处理(方法参照艾德莱琼脂糖凝胶试剂盒),将酶切后的目的基因及pTRV2载体用T4DNA连接酶在室温下连接30min,然后转化至大肠杆菌DH5α中,经菌落PCR鉴定后提取阳性质粒进行测序验证,测序比对结果如图2。将测序成功的重组质粒命名为pTRV2-MsPDS。
2、质粒转化农杆菌
将pTRV-1、pTRV2、pTRV2-MsPDS质粒各1μg(5~10μL)加到100μL农杆菌GV3101感受态细胞中,冰浴5min,液氮速冻5min,37℃水浴5min,冰浴5min后,加200μL无抗生素的液体LB,28℃,150r/min振荡培养2~3h。然后将200μL菌液均匀涂于含kan,rif抗性LB平板上,28℃培养2天。挑单菌落接种到1mL相应抗性液体LB中,28℃震荡培养过夜。PCR鉴定阳性克隆菌液后,如图3所示,保存甘油菌,或者直接接种至新鲜LB中,准备侵染。
3、侵染植株
3.1材料准备
将处理过的紫花苜蓿种子点播在装有基质(营养土:蛭石=1:1(v/v))的花盆中,上面再覆盖一层基质,浇足水后放置于光照培养箱中,培养条件为光照16h,26℃,黑暗8h,22℃。
3.2侵染菌液准备
取100μl pTRV2、pTRV2-MsPDS的农杆菌菌液加入含有50mg/L kana,50mg/Lrif的1mlYEP中,取200μl pTRV1加入2ml含有相应抗生素的YEP中,28℃150rpm过夜培养。将活化后的pTRV2、pTRV2-MsPDS的农杆菌菌液加入相同抗生素浓度的25ml YEP中,pTRV1加入50mlYEP相同抗生素中,28℃150rpm振荡过夜.待菌液OD值达1.5-2.0后,25℃4000rpm离心20min后,倒掉上清液,加入一定体积的重悬液(10mM MgCl2,10mM MES以及200uM乙酰丁香酮)对菌体重悬,黑暗条件下25℃150rpm放置3h。按照1:1的比例将pTRV1分别于pTRV2、pTRV2-MsPDS混匀后用于后续侵染。
4、建立侵染体系
将苗龄处于第4叶期的中苜1号进行侵染,浸泡在装有pTRV1+pTRV2-MsPDS菌液的50mL离心管中,通过真空渗透法将离心管至于真空泵中,0.08Mpa压强下处理20min,然后将其取出移栽至苗钵中继续生长。
观察发现,在侵染10-14d后叶片出现光漂白现象,漂白面积达30%,沉默效率提高了3倍以上,如图4所示。
试验例1
使用‘中苜1号’和‘公农1号’两个紫花苜蓿品种,并根据苗龄和侵染时间设置试验处理,具体试验设计采用完全随机设计,如表1所示。
表1紫花苜蓿VIGS沉默体系建立的试验设计
将长势一致、苗龄处于1至6叶期的紫花苜蓿‘中苜1号’和‘公农1号’应用本发明提供的沉默体系进行侵染,取不同植株分别浸泡在装有pTRV1+pTRV2(对照)和pTRV1+pTRV2-MsPDS菌液的50mL离心管中,通过真空渗透法将离心管至于真空泵中,0.08Mpa压强下处理不同时间(10min、15min、20min、25min),然后将其取出移栽至苗钵中。在侵染10-14d后叶片出现光漂白现象(图4),统计每个处理后沉默效率,统计结果如表2所示,表型结果如图5所示:
表2不同品种的紫花苜蓿在不同发育时期和侵染时间处理下的侵染效果
结果发现:
侵染紫花苜蓿‘中苜1号’的植株时,当侵染时间为10min苗龄处于2叶期时,叶片出现光漂白的现象,侵染效率在10%;当侵染时间为20min时,苗龄在2叶、4叶和5叶期时均出现光漂白现象,侵染效率分别是10%、30%和10%。
侵染紫花苜蓿‘公农1号’的植株时,当侵染时间为10min苗龄处于2叶期时,叶片出现光漂白的现象且效率在10%;当侵染时间为20min时,苗龄在4叶和5叶期时均出现光漂白现象,侵染效率分别是20%和10%。
可见,本发明提供的沉默体系在不同品种中均可以实现MsPDS基因的鉴定,而且对苗龄要求及侵染时间要求低,苗龄在2叶~5叶期,侵染时间在10-20min均可以出现光漂白现象,侵染效率最高可以达到30%。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
序列表
<110> 中国农业大学
<120> 鉴定MsPDS基因的VIGS沉默体系及鉴定方法
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 404
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcgatttgat tttcccgaag tccttccatc tccgttaaat ggaatatggg caatcttgag 60
gaataatgag atgctgacct ggccagagaa aatcaaattt gcaattggac ttcttccagc 120
tatgcttggt ggacaggcgt atgttgaggc tcaagatggt atttctgtca aagaatggat 180
gagaaaacag ggcattcctg aacgtgtaac tgatgaagtg ttcatagcaa tgtcaaaggc 240
cctaaacttc atcaaccctg atgaactttc aatgcaatgt attttgattg ctttaaaccg 300
atttcttcag gagaagcatg gttctaagat ggcctttttg gacggaaacc cccctgaaag 360
actttgtatg ccaattgttg atcatattaa gtccttgggc ggtg 404
<210> 2
<211> 26
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 2
gaattctcga tttgattttc ccgaag 26
<210> 3
<211> 26
<212> DNA/RNA
<213> 人工序列(Artificial Sequence)
<400> 3
ggatcccacc gcccaaggac ttaata 26
Claims (2)
1.鉴定MsPDS基因的方法,其特征在于,包括:
将生长至第四片真叶的紫花苜蓿植株置于鉴定MsPDS基因的VIGS沉默体系中,并在真空压强为0.08Mpa的条件下侵染;
侵染20min后,将其取出移栽至苗钵中使其继续生长,并观察叶片颜色变化;
其中,所述鉴定MsPDS基因的VIGS沉默体系是:
将SEQ ID NO.1所示的核苷酸序列或应用SEQ ID NO.2-3所示的核苷酸序列扩增紫花苜蓿的cDNA得到的目的基因插入到TRV2载体上得到的重组载体。
2.如权利要求1所述的方法,其特征在于,鉴定MsPDS基因的VIGS沉默体系是通过以下步骤获得:
应用SEQ ID NO.2-3所示的碱基序列对紫花苜蓿的cDNA扩增,得到目的基因片段;
使用限制性内切酶EcoRI、BamHI同时对目的基因和VIGS沉默载体进行双酶切,连接后转化大肠杆菌DH5α,并将筛选的阳性菌落转化到农杆菌GV3101,得到鉴定MsPDS基因的VIGS沉默体系。
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