CN117051022A - 一种小麦TaLAC129基因及其编码蛋白质和应用 - Google Patents
一种小麦TaLAC129基因及其编码蛋白质和应用 Download PDFInfo
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Abstract
本发明提供了一种小麦TaLAC129基因,所述小麦TaLAC129基因的核苷酸序列如SEQ ID NO.1所示;编码蛋白质的氨基酸序列如SEQ ID NO.2所示。本发明还提供了所述小麦TaLAC129基因在培育转基因小麦植株中的应用,所述培育转基因小麦植株方法为:将TaLAC129基因导入野生型小麦植株中,使TaLAC129基因过表达,得到转基因植物;研究发现过表达TaLAC129基因能够明显提高小麦的产量,并且能够提高根系漆酶活性和木质素含量,抵抗有害细菌或真菌的侵入,因此,TaLAC129基因可用于提高产量和调节植物抗逆性,对植物育种具有重大的应用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及一种小麦TaLAC129基因及其编码蛋白质和应用。
背景技术
小麦(TriticumaestivumL.)是全世界广泛栽培和种植的粮食作物,全球约40%的人口以小麦为主要口粮。在中国,小麦的播种面积大约占到全国耕地面积的20%~30%,在农业生产中尤为重要。因此,小麦的生产是维持我国民生计划、保障经济社会发展和社会稳定的重要保障。随着城镇化、土地沙漠化和盐碱化,尤其是在耕地资源有限且不断萎缩的情况下,小麦种植面积已不可能大幅度恢复性增长且日益减少,这与粮食消费需求不断刚性增长的矛盾越来越突出和严重。因此,提高小麦单产是确保小麦总产水平提高,同时也是应对日益加剧的粮食危机的终南捷径。
漆酶(LAC,EC1.10.3.2)属于铜蓝蛋白氧化酶家族,是一种多功能含铜氧化酶,LAC在植物生长发育中发挥着不可替代的作用,是形成植物次生细胞壁的关键因素。它调节木质素的沉积,影响维管束的发育,并广泛参与叶、根、花和种子等器官的发育。LAC通过调节木质素的合成影响细胞壁的生长、组织的发育和机械强度,并且会影响植物生殖生长,与作物生产力性状密切相关。
发明内容
本发明所要解决的技术问题在于针对上述现有技术的不足,提供一种小麦TaLAC129基因及其编码蛋白质和应用,将该基因导入目的植株,使TaLAC129基因过表达,得到转基因植株,不仅能够明显提高植物的产量,而且能够提高根系漆酶活性和木质素含量,抵抗有害细菌或者真菌的侵入,可用于调节植物抗逆性。
为解决上述技术问题,本发明采用的技术方案是:一种小麦TaLAC129基因,所述小麦TaLAC129基因的核苷酸序列如SEQ ID NO.1所示,所述小麦TaLAC129基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种包含所述小麦TaLAC129基因的生物材料,所述生物材料包括重组表达载体和工程菌。
本发明还提供了一种所述小麦TaLAC129基因的应用,所述小麦TaLAC129基因用于培育转基因小麦植株;
优选地,所述培育转基因小麦植株的方法为:将所述TaLAC129基因导入野生型小麦植株中,使TaLAC129基因过表达,得到转基因小麦植株;
优选地,所述TaLAC129基因在小麦植株中过表达的方法为:
S1、TaLAC129基因过表达载体的构建
S101、线性化载体:过表达载体BL130035S-Flag经BamHI和XbaI双酶切,得到线性化载体;
S102、获得目的片段:根据目的基因TaLAC129的CDS序列,设计包含有酶切位点和载体末端同源序列的特异性引物;以小麦cDNA为模板,利用高保真酶进行PCR扩增,通过凝胶电泳检测并回收,得到TaLAC129基因的目的片段;所述特异性引物为pBL-TaLAC129-BamHI-F和pBL-TaLAC129-XbaI-R;所述引物pBL-TaLAC129-BamHI-F的核苷酸序列如SEQ IDNO.3所示;所述引物pBL-TaLAC129-XbaI-R的核苷酸序列如SEQ ID NO.4所示;
S103、重组反应、重组产物的转化及菌落PCR鉴定,具体操作方法如下:
S10301、重组反应:于冰上配置反应体系,线性化载体1μL,目的片段1μL,5XBuffer2μL,重组酶1μL,ddH2O5μL;使用移液器吸打混匀,离心将反应液收集至管底后37℃反应30min,得到重组产物;
S10302、重组产物转化:在冰上解冻克隆感受态细胞DH5α,取10μL重组产物加入到100μL感受态细胞中,混匀,冰上静置30min;42℃水浴热激45s后,置于冰上冷却2-3min;加入900μLLB培养基,37℃摇菌1h,转速为200-250rpm;然后5000rpm离心5min,弃掉上清;用LB培养基将菌体重悬,用无菌涂布棒涂布在预热至37℃的抗性LB固体培养基平板上,涂匀;37℃培养箱中倒置培养12-16h;
S10303、菌落PCR鉴定:挑取重组反应转化平板上若干个克隆进行菌落PCR鉴定,扩增引物使用载体上的通用测序引物,如果克隆正确,应有2038bp大小的条带出现;所述通用测序引物为35S-promoter和NOS-R;所述引物35S-promoter的核苷酸序列如SEQ ID NO.5所示;所述引物NOS-R的核苷酸序列如SEQ ID NO.6所示;所述菌落PCR反应体系为:菌液1μL,引物35S-promoter1μL,引物NOS-R1μL,酶5μL,加灭菌ddH2O至10μL;所述PCR反应程序为:95℃预变性3min,95℃变性15s,58℃退火15s,72℃延伸3min,35个循环,最后72℃延伸5min;
S104、挑选克隆正确的菌斑进行下一代测序,得到TaLAC129基因过表达载体质粒p35S:TaLAC129-flag;
S2、TaLAC129基因过表达小麦遗传转化
将S1得到的TaLAC129基因过表达载体质粒p35S:TaLAC129-flag转化农杆菌GV3101感受态细胞,在卡那抗生素和利福平抗生素双抗平板上进行涂布培养;挑取农杆菌单克隆进行菌落PCR鉴定,将目的条带大小正确的克隆接种至培养基,摇菌培养12h;将菌液在双抗平板上进行划线培养,再次选择单克隆进行菌落PCR鉴定,将正确的农杆菌单克隆送至转基因平台进行小麦遗传转化,通过农杆菌侵染法将转基因载体整合到小麦幼胚中,受体材料为春小麦Fielder品种,最终得到TaLAC129转基因植株;
S3、TaLAC129转基因阳性植株的鉴定
将S2得到的TaLAC129转基因植株通过bar试纸检测阳性植株,然后进行PCR扩增,引物为Bar-1300-F和Bar-1300-R,测定植株是否包含bar基因;
所述引物Bar-1300-F的核苷酸序列如SEQ ID NO.7所示;
所述引物Bar-1300-R的核苷酸序列如SEQ ID NO.8所示;
若扩增产物有689bp大小的条带,则测定植株包含bar基因,判定为TaLAC129基因过表达,为TaLAC129转基因阳性植株;若扩增产物无上述大小的条带,则测定植株不包含bar基因,判定为阴性植株。
优选地,所述转基因小麦植株满足如下Ⅰ或Ⅱ中的至少一种表型:
Ⅰ:所述转基因小麦植株根系漆酶活性强于所述野生型小麦植株;所述转基因小麦植株根系木质素含量强于所述野生型小麦植株;
Ⅱ:所述转基因小麦植株产量高于所述野生型小麦植株。
本发明还提供了一种所述小麦TaLAC129基因的应用,所述小麦TaLAC129基因用于高产量小麦育种和高抗逆性小麦育种。
本发明与现有技术相比具有以下有益效果:
本发明提供一种小麦TaLAC129基因,将该基因导入目的植株,使TaLAC129基因过表达,得到转基因植株,发现该转基因植株不仅能够明显提高小麦的产量,而且能够提高根系漆酶活性和木质素含量,抵抗真菌的侵入,可用于调节植物抗逆性。将本发明的TaLAC129基因导入目的植株,得到转基因植物新品种,对于植物育种具有重大的应用价值。
下面结合附图和实施例对本发明作进一步详细说明。
附图说明
图1是本发明实施例2的TaLAC129基因表达载体图谱。
图2是本发明实施例3的转基因小麦株系OE7和OE8的bar基因检测凝胶电泳图。
图3是本发明实施例5的转基因小麦OE7、OE8和野生型小麦(WT)的株高、表型及生物量分析,其中,图a是不同生长期的株高对比;图b是生长表型图对比;图c是不同器官(茎叶、颖壳、籽粒和根系)在收获期的生物量对比。
图4是本发明实施例5的转基因小麦OE7、OE8和野生型小麦(WT)的籽粒性状分析图,其中,图a是粒长表型图,图b是粒宽表型图。
图5是本发明实施例6的转基因小麦OE7、OE8和野生型小麦(WT)的根系漆酶活性和木质素含量分析图。
具体实施方式
实施例1
本实施例提供了TaLAC129基因的克隆方法,包括以下步骤:
1、利用天根生化科技(北京)有限公司的RNAprepPure植物总RNA提取试剂盒(DP432)提取品种“中国春”小麦根系样本的总RNA,用NanodropOneC测定仪检测RNA的纯度和浓度后,-80℃保存。
2、扩增TaLAC129基因全长序列
对提取的RNA进行稀释,然后采用天根公司的FastKing一步法除基因组cDNA第一链合成预混试剂盒(KR118)将RNA反转录成cDNA,以该cDNA作为PCR扩增模板,用引物pBL-TaLAC129-BamHI-F和pBL-TaLAC129-XbaI-R进行PCR扩增;
所述引物pBL-TaLAC129-BamHI-F的核苷酸序列如SEQ ID NO.3所示;所述引物pBL-TaLAC129-XbaI-R的核苷酸序列如SEQ ID NO.4所示;
所述PCR扩增反应体系为:cDNA模板1μL,引物pBL-TaLAC129-BamHI-F1μL,引物pBL-TaLAC129-XbaI-R1μL,高保真酶12.5μL,加灭菌ddH2O至25μL;
所述PCR反应程序为:95℃预变性3min,95℃变性15s,58℃退火15s,72℃延伸2min,35个循环,最后72℃延伸5min。
将获得的产物连接到大肠杆菌,经PCR和酶切鉴定后进行序列测定,得到TaLAC129基因的核苷酸序列如SEQ ID NO.1所示,产物大小为1725bp;编码蛋白质的氨基酸序列如SEQ ID NO.2所示。
实施例2
本实施例为TaLAC129基因表达载体的构建,包括以下步骤:
S1、线性化载体:过表达载体BL130035S-Flag经BamHI和XbaI双酶切,得到线性化载体;
S2、获得目的片段:根据目的基因TaLAC129的CDS序列,设计包含有酶切位点和载体末端同源序列的特异性引物;以“中国春”小麦cDNA为模板,利用高保真酶进行PCR扩增,通过凝胶电泳检测并回收,得到TaLAC129基因的目的片段;所述特异性引物为pBL-TaLAC129-BamHI-F和pBL-TaLAC129-XbaI-R;所述引物pBL-TaLAC129-BamHI-F的核苷酸序列如SEQ ID NO.3所示;所述引物pBL-TaLAC129-XbaI-R的核苷酸序列如SEQ ID NO.4所示;
S3、重组反应、重组产物的转化及菌落PCR鉴定参照诺唯赞C112重组试剂盒说明书进行,具体操作方法如下:
重组反应:于冰上配置反应体系,线性化载体1μL,目的片段1μL,5XBuffer2μL,重组酶1μL,ddH2O5μL;使用移液器轻轻吸打混匀,离心将反应液收集至管底后37℃反应30min,得到重组产物;
重组产物转化:在冰上解冻克隆感受态细胞DH5α,取10μL重组产物加入到100μL感受态细胞中,轻弹管壁混匀,冰上静置30min;42℃水浴热激45s后,立即置于冰上冷却2-3min;加入900μLLB培养基(不添加抗生素),37℃摇菌1h,转速为200-250rpm;然后5000rpm(2400×g)离心5min,弃掉上清,用LB培养基将菌体重悬;将相应抗性的LB固体培养基平板在37℃培养箱中预热,然后用无菌涂布棒将菌液涂布在LB固体培养基平板上,轻轻涂匀;37℃培养箱中倒置培养12-16h;
菌落PCR鉴定:挑取重组反应转化平板上若干个克隆进行菌落PCR鉴定,扩增引物使用载体上的通用测序引物,如果克隆正确,应有2038bp大小的条带出现;所述通用测序引物为35S-promoter和NOS-R;所述引物35S-promoter的核苷酸序列如SEQ ID NO.5所示;所述引物NOS-R的核苷酸序列如SEQ ID NO.6所示;所述菌落PCR反应体系为:菌液1μL,引物35S-promoter1μL,引物NOS-R1μL,酶5μL,加灭菌ddH2O至10μL;所述PCR反应程序为:95℃预变性3min,95℃变性15s,58℃退火15s,72℃延伸3min,35个循环,最后72℃延伸5min;
S4、挑选克隆正确的菌斑进行下一代测序,得到TaLAC129基因过表达载体质粒p35S:TaLAC129-flag。
图1是本实施例的TaLAC129基因表达载体图谱。
实施例3
本实施例为TaLAC129基因过表达小麦遗传转化。
将测序正确的过表达载体质粒,通过“冻融法”转化进农杆菌(GV3101)感受态细胞,在双抗(卡那抗生素和利福平抗生素)平板上进行涂布培养。挑取单克隆进行菌落PCR鉴定,鉴定的方法及扩增程序同实施例2,将目的条带大小正确的克隆接种至培养基,摇菌培养12h。将菌液在双抗平板上进行划线培养,再次选择单克隆进行菌落PCR鉴定,将正确的农杆菌单克隆送至转基因平台进行小麦遗传转化,通过农杆菌侵染法将转基因载体整合到小麦幼胚中,受体材料为春小麦Fielder品种,最终得到TaLAC129转基因植株。
通过转基因平台共获得22棵转基因T0幼苗,将T0幼苗继续种植培养得到T1代植株,然后使用bar检测试纸进行阳性苗检测;然后将鉴定出的T1代阳性植株继续培养得到T2代植株,共17棵,DNA提取小麦植株bar基因进行阳性植株确认检测,PCR扩增引物为Bar-1300-F和Bar-1300-R;
所述引物Bar-1300-F的核苷酸序列如SEQ ID NO.7所示;
所述引物Bar-1300-R的核苷酸序列如SEQ ID NO.8所示;
若扩增产物有689bp大小的条带,则测定植株包含bar基因,判定为TaLAC129基因过表达,为TaLAC129转基因阳性植株;若扩增产物无上述大小的条带,则测定植株不包含bar基因,判定为阴性植株。
结果显示,本实施例的TaLAC129基因过表达小麦遗传转化得到两个阳性转基因小麦株系OE7和OE8,OE7和OE8株系的bar基因检测凝胶电泳图见图2,图中M泳道为Marker,1-17分别为小麦植株的PCR样本。
实施例4
本实施例为TaLAC129基因过表达小麦的盆栽试验。
1、试验设计
将野生型春小麦Fielder品种(WT)和筛选后得到的两个阳性转基因小麦株系(OE7和OE8)种植在花盆中,每盆装入2.5kg土壤,每盆两株小麦幼苗。于种植前在花盆土壤中施入按纯N0.2g·kg-1土壤计的尿素作为小麦生长底肥。试验设计在小麦生长的收获期进行采样,设置四个重复。
盆栽试验用土取自陕西杨陵区马家底村田间土,多点随机取土混匀待用。土壤类型为塿土,基本理化性质如表1所示:
表1土壤类理化性质
2、样品采集与测定
在小麦收获期测定转基因植株和野生型植株每盆两株小麦的株高,人工记录盆栽内小麦的穗长、穗数。完整收获各处理每盆小麦地上部植株样品,晒干、脱粒后分别测定茎叶、颖壳生物量以及产量(籽粒生物量)、粒长、粒宽、穗粒数和千粒重。
实施例5
本实施例为过表达TaLAC129基因对小麦产量的影响分析。
图3是本发明实施例5的转基因小麦OE7、OE8和野生型小麦(WT)的株高、表型及生物量分析,其中,图3a是不同生长期的株高对比;图3b是生长表型图对比;图3c是不同器官(茎叶、颖壳、籽粒和根系)在收获期的生物量对比。由图3a可知,OE7和OE8两个转基因小麦株系的株高在拔节期和抽穗期均没有显著差异;而过表达TaLAC129基因收获期小麦的株高显著增加(P<0.05),和WT相比,OE7株系的株高增加了6.8%,OE8植株的株高增加了8.6%。由图3c小麦不同器官(茎叶、颖壳、籽粒和根系)在收获期的生物量对比,发现过表达TaLAC129基因可以一定程度降低小麦茎叶的生物量,但是和WT相比,只有OE7株系达到显著水平(P<0.05);而不同株系之间颖壳生物量没有显著差异;与WT相比,转基因小麦的籽粒生物量(籽粒产量)显著增加,OE7和OE8分别增加了92.72%和61.26%;而转基因小麦的根系生物量却显著降低,OE7和OE8分别降低了53.40%和41.93%。
图4是转基因小麦OE7、OE8和野生型小麦(WT)的籽粒性状分析图,其中,图a是粒长表型图,图b是粒宽表型图,bar=2cm。由图可知,过表达TaLAC129基因显著降低了小麦籽粒长度,却增加了籽粒宽度,转基因小麦株系的籽粒更为饱满;其中,粒长表现为WT>OE8>OE7;粒宽表现为WT<OE8<OE7。
小麦收获后测定了和籽粒相关的农艺性状,包括穗数、穗粒数、千粒重、穗长、粒长和粒宽,并计算了收获指数HI(见表2)。结果表明,与WT相比,转基因小麦株系的穗数和穗长没有差异显著性,但是有下降趋势;转基因小麦株系的穗粒数均有不同程度增加,其中株系OE7具有显著性差异,其穗粒数平均为27.83;千粒重也显著增加(P<0.05),OE7和OE8比WT分别增加了55.72%和36.93%;与WT相比,过表达TaLAC129提高了小麦收获指数,其中OE7具有显著差异,其收获指数平均为0.41。
表2转基因小麦和野生型小麦的籽粒相关的农艺性状
以上结果表明,过表达TaLAC129基因使得小麦的粒宽增加,籽粒更饱满,对于产量三要素中的穗粒数和千粒重均有影响,使其显著增加,因此,过表达TaLAC129转基因植株可以有效提高小麦产量。
实施例6
本实施例为过表达TaLAC129基因对小麦抗逆性的影响分析,包括根系漆酶活性和木质素含量分析。测定方法如下:
漆酶活性的测定:采用可见分光光度法并根据试剂盒(BC1630)说明书进行测定,称取不同采样时期小麦根系0.1g,加入1mL提取液,进行冰浴匀浆;10000g4℃离心10min,取上清,置冰上待测;将分光光度计预热30min以上,调节波长至420nm,用蒸馏水调零;在1mL玻璃比色皿中按照测定管中样本150μL,试剂盒工作液850μL;空白管中蒸馏水150μL,试剂盒工作液850μL分别加入试剂,充分混匀后于420nm处测定10s时的吸光值A1,迅速置于45℃水浴3min,拿出迅速擦干测定190s时的吸光值A2,计算ΔA测定管=A2测定-A1测定,ΔA空白管=A2空白-A1空白,ΔA=ΔA测定管-ΔA空白管。根据公式LAC酶活(U·g-1)=61.7×ΔA÷W进行计算,其中W为根系质量0.1g。
木质素含量的测定:采用紫外分光光度法并根据试剂盒(BC4200)说明书进行测定,称取5mg经80℃烘干至恒重,并经研磨过30-50目筛的根系样品于2mLEP管中;分光光度计预热30min以上,调节波长至280nm,用冰乙酸调零;接下来进行乙酰化:(1)同时将测定管(样本5mg+试剂一500μL)和空白管65℃水浴反应30min;然后3000g室温离心5min,去上清,留沉淀;(2)接上一步再分别加入500μL试剂二,然后涡旋震荡5min,3000g室温离心5min,去上清,留沉淀;(3)接上一步再分别加入500μL试剂三,然后涡旋震荡5min,3000g室温离心5min,去上清,留沉淀;(4)分别再加入500μL试剂四和20μL试剂五,充分混匀(封口膜密封,防止水分散失),于80℃水浴锅水浴40min,进行乙酰化,每隔10min震荡30s,然后自然冷却至室温;(5)最后再分别加入500μL试剂六,充分混匀,于8000g室温离心10min,取上清待测。测定:分别在测定管和空白管中取上一步上清液20μL和冰乙酸980μL,进行充分混匀,吸取反应液于1mL石英比色皿中,测定280nm下的吸光值A,记为A测定管、A空白管,计算ΔA=A测定管-A空白管。空白管只测1-2次。根据公式进行计算:木质素含量(mg·g-1)=2.184×ΔA÷W,其中W为根系质量5mg。
测定结果如图5所示,由图5a可知,过表达TaLAC129可以一定程度上提高小麦根内的LAC活性;在小麦生长60d时,OE7和OE8两个过表达株系和WT相比较,根内LAC活性分别增加了29.03%和53.92%(P>0.05);在小麦生长90d时,同样将OE7和OE8两个过表达株系和WT相比较,根内LAC活性分别显著增加了39.67%和48.35%(P<0.05);随着小麦的生长,90d时的根系LAC活性整体低于60d时根系的LAC活性。由图5b可知,过表达TaLAC129可以提高生长90d小麦根系的木质素含量,整体变化趋势和根内LAC活性一致。在小麦生长60d时,OE7和OE8两个过表达株系和WT相比较,根系木质素含量分别增加了7.91%和16.12%。在小麦生长90d时,同样将OE7和OE8两个过表达株系和WT相比较,根系木质素含量分别显著增加了16.38%和21.67%(P<0.05);同样地,随着小麦的生长,在小麦生长90d时的根系木质素含量整体低于生长60d时根系的木质素含量。结果表明,过表达TaLAC129基因能够提高小麦根系漆酶活性和木质素含量;结合以往的研究可以发现,根系木质素含量的提高,可以抵抗有害病原菌的侵入,可用于提高植物抗逆性。
以上所述,仅是本发明的较佳实施例,并非对本发明作任何限制。凡是根据发明技术实质对以上实施例所作的任何简单修改、变更以及等效变化,均仍属于本发明技术方案的保护范围内。
Claims (7)
1.一种小麦TaLAC129基因,其特征在于,所述小麦TaLAC129基因的核苷酸序列如SEQID NO.1所示,所述小麦TaLAC129基因编码的蛋白质的氨基酸序列如SEQ ID NO.2所示。
2.一种如权利要求1所述的小麦TaLAC129基因的应用,其特征在于,所述小麦TaLAC129基因用于培育转基因小麦植株。
3.根据权利要求2所述的应用,其特征在于,所述培育转基因小麦植株的方法为:将所述TaLAC129基因导入野生型小麦植株中,使TaLAC129基因过表达,得到转基因小麦植株。
4.根据权利要求3所述的应用,其特征在于,所述TaLAC129基因在小麦植株中过表达的方法为:
S1、TaLAC129基因过表达载体的构建
S101、线性化载体:过表达载体BL130035S-Flag经BamHI和XbaI双酶切,得到线性化载体;
S102、获得目的片段:根据目的基因TaLAC129的CDS序列,设计包含有酶切位点和载体末端同源序列的特异性引物;以小麦cDNA为模板,利用高保真酶进行PCR扩增,通过凝胶电泳检测并回收,得到TaLAC129基因的目的片段;所述特异性引物为pBL-TaLAC129-BamHI-F和pBL-TaLAC129-XbaI-R;所述引物pBL-TaLAC129-BamHI-F的核苷酸序列如SEQ ID NO.3所示;所述引物pBL-TaLAC129-XbaI-R的核苷酸序列如SEQ ID NO.4所示;
S103、重组反应、重组产物的转化及菌落PCR鉴定,具体操作方法如下:
S10301、重组反应:于冰上配置反应体系,线性化载体1μL,目的片段1μL,5X Buffer 2μL,重组酶1μL,ddH2O 5μL;使用移液器吸打混匀,离心将反应液收集至管底后37℃反应30min,得到重组产物;
S10302、重组产物转化:在冰上解冻克隆感受态细胞DH5α,取10μLS10301中得到的重组产物加入到100μL感受态细胞中,混匀,冰上静置30min;42℃水浴热激45s后,置于冰上冷却2-3min;加入900μL LB培养基,37℃摇菌1h,转速为200-250rpm;然后5000rpm离心5min,弃掉上清;用LB培养基将菌体重悬,用无菌涂布棒涂布在预热至37℃的抗性LB固体培养基平板上,涂匀;37℃培养箱中倒置培养12-16h;
S10303、菌落PCR鉴定:挑取重组反应转化平板上若干个克隆进行菌落PCR鉴定,扩增引物使用载体上的通用测序引物,如果克隆正确,应有2038bp大小的条带出现;所述通用测序引物为35S-promoter和NOS-R;所述引物35S-promoter的核苷酸序列如SEQ ID NO.5所示;所述引物NOS-R的核苷酸序列如SEQ ID NO.6所示;所述菌落PCR反应体系为:菌液1μL,引物35S-promoter 1μL,引物NOS-R 1μL,酶5μL,加灭菌ddH2O至10μL;所述PCR反应程序为:95℃预变性3min,95℃变性15s,58℃退火15s,72℃延伸3min,35个循环,最后72℃延伸5min;
S104、挑选克隆正确的菌斑进行下一代测序,得到TaLAC129基因过表达载体质粒p35S:TaLAC129-flag;
S2、TaLAC129基因过表达小麦遗传转化
将S1得到的TaLAC129基因过表达载体质粒p35S:TaLAC129-flag转化农杆菌GV3101感受态细胞,在卡那抗生素和利福平抗生素双抗平板上进行涂布培养;挑取农杆菌单克隆进行菌落PCR鉴定,将目的条带大小正确的克隆接种至培养基,摇菌培养12h;将菌液在双抗平板上进行划线培养,再次选择单克隆进行菌落PCR鉴定,将正确的农杆菌单克隆送至转基因平台进行小麦遗传转化,通过农杆菌侵染法将转基因载体整合到小麦幼胚中,受体材料为春小麦Fielder品种,最终得到TaLAC129转基因植株;
S3、TaLAC129转基因阳性植株的鉴定
将S2得到的TaLAC129转基因植株通过bar试纸检测阳性植株,然后进行PCR扩增,引物为Bar-1300-F和Bar-1300-R,测定植株是否包含bar基因;
所述引物Bar-1300-F的核苷酸序列如SEQ ID NO.7所示;
所述引物Bar-1300-R的核苷酸序列如SEQ ID NO.8所示;
若扩增产物有689bp大小的条带,则测定植株包含bar基因,判定为TaLAC129基因过表达,为TaLAC129转基因阳性植株;若扩增产物无上述大小的条带,则测定植株不包含bar基因,判定为阴性植株。
5.根据权利要求3所述的应用,其特征在于,所述转基因小麦植株满足如下Ⅰ或Ⅱ中的至少一种表型:
Ⅰ:所述转基因小麦植株根系漆酶活性强于所述野生型小麦植株;所述转基因小麦植株根系木质素含量高于所述野生型小麦植株;
Ⅱ:所述转基因小麦植株产量高于所述野生型小麦植株。
6.一种如权利要求1所述的小麦TaLAC129基因的应用,其特征在于,所述小麦TaLAC129基因用于高产量小麦育种。
7.一种如权利要求1所述的小麦TaLAC129基因的应用,其特征在于,所述小麦TaLAC129基因用于高抗逆性小麦育种。
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CN117802114A (zh) * | 2024-01-29 | 2024-04-02 | 云南省农业科学院粮食作物研究所 | Zm00001d042906基因在调控玉米穗长中的应用 |
CN117646029B (zh) * | 2023-11-21 | 2024-07-16 | 贵州大学 | TaSLC25A4-7A基因在小麦育种及提高抗逆能力中的应用 |
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CN117802114A (zh) * | 2024-01-29 | 2024-04-02 | 云南省农业科学院粮食作物研究所 | Zm00001d042906基因在调控玉米穗长中的应用 |
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