CN104327173B - 一种调控植物耐盐性的棉花WRKY转录因子GarWRKY22及应用 - Google Patents
一种调控植物耐盐性的棉花WRKY转录因子GarWRKY22及应用 Download PDFInfo
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Abstract
本发明公开了一种棉花耐盐相关转录因子,即棉属野生种旱地棉WRKY基因GarWRKY22,具有序列表中SEQ ID N0.2的序列。本发明利用电子克隆及RT‑PCR技术分离了一个棉花耐逆性相关蛋白GarWRKY22,通过转化拟南芥进行功能验证,证明所述该转录因子能够调控耐盐性,过表达该基因,植株抗盐能力明显降低。
Description
技术领域
本发明属于生物基因工程技术领域,是一种负调控植物耐盐性的棉花转录因子GarWRKY22。
背景技术
土壤盐渍化是影响农业生产的一个世界性问题,它是影响农业产量和导致农业欠收的一个重要因素。全世界盐渍土面积约10亿hm2,我国地域辽阔,海岸线长,既有大量的滨海盐碱地,又有内陆盐碱地,我国盐渍土面积总计约1亿hm2,有670万公顷属盐碱土耕地,有2千万公顷盐碱荒地尚待开发,而且这个数据仍在上升,另外北方灌溉地区的次生盐渍化土地生产潜力还有待进一步挖掘。江苏滨海滩涂地约为60多万hm2,其中只有少部分改良利用,绝大部分仍未脱盐及不断遭受盐渍危害。面对人口增长,工业发展,耕地面积逐渐减少的趋势,盐碱地的改良和利用已经得到各方面的极大重视;一直以来,有关盐渍土的开发利用主要采取两种措施:一是通过工程措施来改良盐渍土,如通过暗管排水处理等方法;二是开展生物治理,即通过农业生物技术培育耐盐植物品种或开发利用有经济价值的盐生植物资源以改良盐渍土。前者虽然取得了一定效果,但耗资巨大,且治理效果难以长久保持;而后者已成为改良盐渍土的研究热点,耐盐作物品种的选育和利用将是盐碱地改良的一个基本方向。棉花属中等耐盐作物,是盐碱地先锋作物。近年来,随着人口增加、耕地面积减少,粮棉油安全供给的矛盾日益突出。进一步发掘棉花抗胁迫能力,将棉花生产向沿海滩涂及内陆盐碱地转移,对缓解粮棉争地矛盾,稳定植棉面积,实现农业生产的可持续发展具有十分重要的意义。
植物WRKY转录因子是近年来新发现的植物特有新型锌指型转录调控因子。WRKY通过特异结合靶基因启动子中的W盒(T)(T)TGAC(C/T),来调控靶基因的转录水平,参与植物对非生物胁迫的应答,最终使植物体内某种胁迫达到平衡。目前,在多种植物中都已发现WRKY转录因子家族成员,并在一些模式植物的研究中解析了该转录因子在植物应答逆境胁迫的功能和调控网络,棉花中也发现了不少WRKY转录因子,但棉花中的与耐盐相关的WRKY转录因子的研究鲜少报道。
发明内容
本发明提供一种调控植物耐盐性的棉花WRKY转录因子基因GarWRKY22,所述基因cDNA核苷酸序列如SEQ ID NO.2所示。
所述的调控植物耐盐性的棉花GarWRKY22基因编码的蛋白质,具有序列表中SEQID NO.3所述的氨基酸序列。
本发明提供了含有上述棉花WRKY转录因子GarWRKY的植物表达载体pCAMBIA2301。将GarWRKY22基因克隆到pCAMBIA2301,获得pCAMBIA2301-CaMV35S-GarWRKY22。
本发明所述基因GarWRKY22在培育耐盐植物中的应用。具体是将GarWRKY22基因通过植物表达载体转入目的植物内。所述植物优选是模式植物拟南芥。
本发明的有益效果:利用现有的植物基因工程技术,利用电子克隆及RT-PCR技术,分离与鉴定棉花耐盐相关基因序列信息,并通过根癌农杆菌浸花法将基因转入拟南芥,经过耐盐表型鉴定证明转基因植株的耐盐力明显降低。
附图说明
图1GarWRKY22基因全长cDNA序列的扩增结果。
图2实时荧光定量PCR法(qRT-PCR)分析盐处理不同时期GarWRKY22基因
在根中的表达情况。
图3实时荧光定量PCR法(qRT-PCR)分析盐处理不同时期GarWRKY22基因
在叶片中的表达情况。
图4植物表达载体pCAMBIA2301-CaMV35S-GarWRKY22的构建
(a)大肠杆菌pCAMBIA2301-CaMV35S-GarWRKY 22PCR检测电泳结果
(b)pCAMBIA2301-CaMV35S-GarWRKY22的酶切鉴定。
图5转基因植株的PCR鉴定
PCR扩增载体pCAMBIA2301-CaMV35S-GarWRKY22抗性拟南芥目的基因。
图6转基因烟草及野生型对照在不同浓度NaCl处理下的生长
生长在含不同浓度NaCl的MS培养基上。
图7根系长度的测量NaCl条件下,正常培养一周的根长表现。此图表示0mM、100mM150mM NaCl条件下野生型和转基因株系萌发率的统计分析。
具体实施方式
实施例1、GarWRKY22基因的获得
1.1RNA的提取
提取RNA
(1)取0.5g新鲜棉花组织,加入0.1g交联聚乙烯砒咯烷酮(PVPP),在液氮中充分研磨至粉末,将冻粉末迅速转入10ml离心管中,加5ml CTAB提取液与500μL0.1M pH8.0的Tris-HCl,65℃水浴20min,中途翻转混匀;
(2)加等体积氯仿充分混匀,冰浴静置10min;
(3)4℃,10000rpm离心20min。分装于4个1.5ml离心管;
(4)吸上清,加入1/3体积的8M LiCl混匀,-70℃30min或-20℃过夜;
(5)4℃,10000rpm离心20min。弃上清,70%乙醇洗涤两次后,吹干沉淀溶解于30μLDEPC水;
(6)加入10U无RNase活性的DNase和25U RNase Inhabitor,10×buffer消化30min后加等体积氯仿,抽提一次;
(7)上清转移到新管中,加1/10体积的3M pH 5.2NaAc和等体积的异丙醇或2.5倍体积的无水乙醇,-20℃放置过夜或-70℃冰浴3h;
(8)4℃,10000rpm离心20min,弃上清,70%乙醇洗涤两次后溶解于30μL DEPC水。即得棉花RNA。
1.2cDNA的合成
体系:
cDNA第一链的合成
65℃(10min)→冰上放置(2min)
M-MLV(反转录酶,takara,200UμL-1) 0.5μL
5×buffer 4μL
42℃(2h)→70℃(10min)→4℃
1.3cDNA克隆
根据本实验室旱地棉转录组测序(Xu et al.,2013)(De novo transcriptomesequencing and comparative analysis of differentially expressed genes inGossypium aridum under salt stress)获得的6个EST序列,序列拼接得到一长为1888pb长的序列,其核酸序列如SEQ ID NO.1所示。用软件ORF Finder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)预测完整ORF。在ORF序列两侧设计上游引物5’-ATGGCTGCTTCATCATCATCTGC-3’(SEQ ID NO.4)和下游引物5’-TCAAGACAGTAATCCGTCCA–3’(SEQ ID NO.5),用旱地棉cDNA(由步骤1.2合成的)进行RT-PCR,得到包含整个编码框的GarWRKY cDNA克隆,全长1530bp(SEQ ID NO.2(图1)。回收该片段,克隆至PTG19-T-载体(北京全式金生物技术有限公司)中鉴定、测序。参照文献cai et al.,2014(Genome-wideanalysis of the WRKY transcription factor gene family in Gossypium raimondiiand the expression of orthologs in cultivated tetraploid cotton),我们将分离得到的旱地棉WRKY基因命名为GarWRKY22,其编码的氨基酸序列如SEQ ID NO.3所示。
1.3.1PCR反应体系(50μL)
1.3.2PCR扩增程序:94℃3min;94℃30sec,60℃30sec,72℃3min,34个循环;72℃10min;4℃保温。
C、扩增得到该基因的ORF序列,回收扩增产物,并克隆到PTG19-T载体,筛选阳性克隆,测序由上海英俊公司完成。
1.4基因表达分析(qRT-PCR)
1.4.1盐胁迫下RNA的提取
材料棉属野生种旱地棉,将旱地棉种子播于盆钵中等待发芽,发芽后约3天待子叶展开,将只有主根的小苗从土中取出,洗去根上的泥土,将苗浸在营养液中培养,待长至4-6片真叶时,用含200mM NaCl溶液(其中CNa+:CCa2+为15:1)的营养液按不同的处理时间(0h、1h、3h、6h、12h、24h、36h、48h、72h)处理苗,分别取叶片、根,提取RNA,方法同上。
1.4.2逆转录(RT)产生cDNA
方法同上。
1.4.3PCR反应
①以cDNA为模板,引物为
GarWRKY22-RT-F:5‘-ACTTGTAATAACTGCGTGG-3’(SEQ ID NO.6)
GarWRKY22-RT-R:5‘-CCAATACCATCAAACATCACAG-3’(SEQ ID NO.7)
②PCR反应体系:
③PCR程序:
将PCR反应的组分别加于qRT-PCR专用96-孔板(Applied Biosystems)中,加盖专用的高透光率封口膜(Applied Biosystems),用Applied Biosystems 7500Fast Real-Time PCR System)进行qRT-PCR,采用两步法PCR扩增标准程序:95℃30sec;95℃5sec,60℃34sec共40个循环;95℃15sec,60℃1min,95℃15sec。
每个样品三次重复。
反应结束后确认扩增曲线和溶解曲线,可以通过溶解曲线分析确认PCR反应大的特异性。计算CT值,结果见图2、3。
实施例2、植物表达载体的构建
2.1pCAMBIA2301-CaMV35S-GarWRKY22植物表达载体的构建
植物表达载体pCAMBIA2301-CaMV35S质粒(冯娟等.,2013;棉属野生种旱地棉蛋白激酶基因GarCIPK8的克隆与功能分析)。选用BamHⅠ和KnpⅠ分别对pCAMBIA2301-CaMV35S和目的基因片段GarWRKY22进行酶切,回收载体大片段和目的基因片段,用T4连接酶连接后转化大肠杆菌Trans1-T1感受态细胞(购自北京全式金生物技术有限公司),鉴定重组子后即得到带有目的基因的植物表达载体。
pCAMBIA2301-CaMV35S质粒和目的基因片段的酶切
质粒双酶切体系如下:
于30℃酶切,反应时间≥5h。双酶切后琼脂糖凝胶对双酶切产物进行电泳检测,结果见图4。
GarWRKY22片段双酶切体系:
30℃酶切过夜
回收pCAMBIA2301-CaMV35S载体大片段和目的基因片段。
2.1.1基因与酶切得到的pCAMBIA2301大片段的连接
连接反应体系:
16℃连接过夜。
2.1.2转化大肠杆菌
连接产物转化大肠杆菌感受态细胞,在含Amp100mg/L的LB液体培养基中37℃振荡培养。
2.1.4重组子的鉴定
①PCR鉴定
②挑取单菌落接种于1.5mL含卡那霉素的LB液体培养基中37℃振荡培养,用GarWRKY22基因特异性引物GarWRKY22F-BamHI:5’-CGCGGATCCGATGGCTGCTTCATCATCATCTGC-3’(SEQ ID NO.8)和GarWRKY22R-KpnI:5'-CGGGGTACCTCAAGACAGTAATCCGTCCA-3'-3(SEQ IDNO.9)进行PCR扩增,琼脂糖凝胶电泳检测是否含有预期片段。PCR反应程序如下::94℃5min;94℃30sec,55℃45sec,72℃1min30sec,36个循环;72℃10min;4℃保温。质粒的酶切鉴定
用碱变性法提取质粒,选取BamHⅠ和KnpⅠ酶进行酶切,酶切体系同上,琼脂糖凝胶电泳检测是否有预期片段。
③pCAMBIA2301-CaMV35S-GarWRKY22提取和保存
挑取①和②鉴定的阳性单菌落接种于5mL含卡娜青霉素的LB液体培养基中37℃过夜振荡培养,用质粒提取试剂盒(AxyPrep Plasmid Miniprep Kit,康宁生命科学有限公司)提取质粒并将质粒保存在-20℃备用。
实施例3、农杆菌感受态的制备与转化
3.1农杆菌EHA105感受态的制备
(1)挑取EHA105单菌落,接种于5ml LB液体培养基中,28℃,200rpm震荡培养过夜至OD600值为0.4;
(2)以1:100接种于400-500ml LB培养基中(1L三角瓶中),摇菌至OD600为0.6-0.8,冰浴10min;
(3)预冷的50ml离心管中收集菌液,4℃,5000rpm,离心5min;
(4)弃上清,沉淀用无菌水充分悬浮,4℃,5000rpm,离心5min;重复此过程3次,。
(5)向洗好的菌体中加1ml(根据菌体多少而定)含10%无菌甘油重悬浮细胞。
(6)分装成50μL每管,液氮速冻,置于-80℃备用。
3.2电击法转化农杆菌感受态细胞EHA105
1.取出农杆菌感受态细胞于冰上冻融。
2.加2μl pCAMBIA2301-CaMV35S-GarWRKY22质粒DNA于50μl感受态细胞中,用枪头轻轻搅拌混匀。
3.取出细胞与质粒的混合物转入电击杯中(电击杯-20℃预冷),吸干电击杯表面的水,将电击杯放入电转化仪的电极之间,在2400V高压下电击4-5s。
4.取出电击杯,迅速加入1ml LB液体培养基不含抗生素),混匀并转移混合液到1.5ml离心管中,28℃,200rpm振荡培养3h。
5.取100ul菌液涂布于含Rif(50mg/L)和Kan(100mg/L)LB平板上,28℃倒置培养2-3天。
注:
1.细胞与质粒的混合物应沿槽壁慢慢加入电击杯中,避免产生气泡。
2.电击前擦干电击杯外侧。
3.涂板菌液量可根据菌液浓度作调整。
3.3菌体PCR鉴定
菌体PCR方法及程序同上(同步骤2.1.4)。
3.4含GarWRKY22基因的农杆菌的保存
挑取3.3鉴定的阳性克隆接种到5ml LB(50mg/L的Rif和100mg/L的Kan)中28℃,200rpm振荡培养1-2天直到OD600=0.6-1.0。然后向灭菌的1.5ml离心管加入灭菌的50%的甘油300μl和菌液700μl,混匀,保存于-80℃的冰箱中备用。
实施例4、转化拟南芥及转基因功能验证
4.1浸花法侵染拟南芥
4.1.1转化液的配置(现配现用)
1/2MS
Sucrose:5%
silwette-775μl/100ml
PH:5.8
4.1.2转化
1.选择盆中长出约20-30个花絮的拟南芥,剪掉已成熟的果荚,转化前2-3天浇足水。
2.用接种环将3.4保存的含GarWRKY22基因的农杆菌接种到1.5ml离心管中28℃,200rpm振荡培养1-2天。
3.取5ul到50ml LB(50mg/L的Rif和100mg/L的Kan)培养基中,28℃,200rpm振荡培养直到OD600=0.6-1.0
4.700g离心5分钟
5.弃上清,收集菌体,用100ml的MS转化液重悬浮菌体备用
6.将待转化的植株倒置于MS转化液中45s(花蕾要全部浸入转化液中)。
7.套袋,暗光培样24h后至于正常培养条件下。
8.一周后再转化一次,步骤同上。
4.2转基因阳性植株的鉴定
4.2.1播种
1.将待播种子装于2ml的EB管中
2.70%的乙醇(v/v)消毒1-3min,无菌水洗一次
3.15%的NaClO(v/v)消毒5min,9000rpm离心2分钟
4.无菌水清洗3-5次
5.加适量的无菌水
6.4℃暗培养2-3天
4.2.2阳性植株的鉴定
1.将收获的T0代种子播于含100mg L–1卡那霉素的1/2MS培养基上,2周后观察,转基因植株长出真叶并呈绿色,表型正常,而非转基因植株生长停留在子叶期,并且呈黄。
2.将长出真叶并呈绿色的拟南芥植株移栽到土质基质上,置于培养箱中正常培养数周
3.采集叶片进行基因组水平和转录组水平的PCR验证,PCR扩增转基因植株的基因组DNA为模板,引物用基因特异性引物GarWRKY22F-BamHI(SEQ ID NO.8),GarWRKY22R-KpnI(SEQ ID NO.9),PCR反应体系和反应条件同上,结果见图5。
4.3拟南芥转化系的抗性鉴定
4.3.1播种
选取T1代拟南芥转基因种子与野生型种子,将消毒的野生型(作为对照)及T1代纯合体种子分别平铺到1/2MS播种培养基上,暗培养2-3d;然后放置18/22℃,16hr光照/8hr黑暗的条件下培养。
4.3.2幼苗耐逆性试验的鉴定
当植株根长为1cm时,选取根长长势基本一致的野生型植株和转基因阳性植株植,分成两组,分别转移至含0mM L-1、100mM L-1浓度NaCl的MS固体培养基上,8/22℃,16hr光照/8hr黑暗的条件下培养,选取转基因植株3个家系做处理,每个处理至少10株苗。继续培养10天后,观察转基因拟南芥和野生型拟南芥幼苗的生长情况。整个生长过程中,MS平板垂直放置。
如图6、7所示,在0mmol L–1NaCl MS培养基上,转基因株系与野生型的根长和生长情况无显著差异,在含100mmol L–1NaCl的MS培养基上转基因植株与野生型植株均表现出盐胁迫伤害,转基因植株主根长显著短于野生型。在200mmol L–1NaCl的MS培养基上,转基因植株较野生型植株表现出盐胁迫伤害且植株明显黄化,且主根长显著短于野生型。
Claims (2)
1.如SEQ ID No.2所示的调控植物耐盐性的棉花转录因子GarWRKY22在培育耐盐植物中的应用。
2.根据权利要求1所述的应用,其特征在于,是将GarWRKY22基因通过植物表达载体转入目的植物内。
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