CN116716316B - 一个响应环境温度调控陆地棉开花的关键基因GhPIF7 - Google Patents
一个响应环境温度调控陆地棉开花的关键基因GhPIF7 Download PDFInfo
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Abstract
本发明公开了一个响应环境温度调控陆地棉开花的关键基因GhPIF7,属于基因工程技术领域,该基因的核苷酸序列如SEQ ID NO:1所示,还公开了该基因在提高棉花抵抗苗期高温胁迫能力中的应用。本发明将目的基因进行克隆,构建目的基因的VIGS沉默载体,研究在苗期相对高温胁迫下的生物学功能,结果发现,GhPIF7基因沉默导致陆地棉的株高变矮、现蕾和开花时间延迟,影响陆地棉的生长发育。以此证实GhPIF7基因正向调控棉花响应高温胁迫反应,改善苗期高温对棉花的开花、现蕾、株高等表型生长发育的影响,为陆地棉耐高温胁迫育种提供重要基因资源。
Description
技术领域
本发明涉及基因工程技术领域,特别是涉及一个响应环境温度调控陆地棉开花的关键基因GhPIF7。
背景技术
棉花是最主要的经济作物之一,在日常生活和工业中扮演着不可或缺的角色。随着全球气候的变暖,温度成为当前作物栽培所考虑的重要原因之一。在种植棉花区域之一的西北内陆地区,苗期时的温度基本持续在20℃左右,然而由于气候变化,使得高温日数增加。一定范围内的高温对棉花的生长发育有促进作用,但是如果温度过高或者较长时间高温,则会影响苗期株高、茎粗、叶数、叶面积、净光合速率和开花时间、铃结率、皮棉产量、籽棉产量和棉纤维质量,导致棉花减产。有研究表明,1985-2021年在伊州区的棉花生长地区气温升高使得高温日数增加,积温增加,导致该区域棉花的播种、出苗、三叶、五叶均不同程度的提前,现蕾、裂铃和吐絮期有程度不一的延迟;致使棉花生长发育受温度的影响较显著。因此,研究苗期相对高温对棉花生长发育的影响是十分重要的。
发明内容
本发明的目的是提供一个响应环境温度调控陆地棉开花的关键基因GhPIF7,以解决上述现有技术存在的问题,本发明证实GhPIF7基因正向调控棉花响应高温胁迫反应,改善苗期高温对棉花的开花、现蕾、株高等表型生长发育的影响,为陆地棉耐高温胁迫育种提供重要基因资源。
为实现上述目的,本发明提供了如下方案:
本发明提供一个响应环境温度调控陆地棉开花的关键基因GhPIF7,其核苷酸序列如SEQ ID NO:1所示。
本发明还提供一种扩增所述关键基因GhPIF7的引物组,所述引物组的核苷酸序列如SEQ ID NO:2-3所示。
本发明还提供一种重组载体,包含所述的关键基因GhPIF7。
本发明还提供一种重组菌,包含所述的重组载体。
本发明还提供一种所述的关键基因GhPIF7或所述的重组载体或所述的重组菌在提高陆地棉抵抗高温胁迫能力中的应用。
本发明还提供一种所述的关键基因GhPIF7或所述的重组载体或所述的重组菌在改善陆地棉响应高温胁迫的生长发育中的应用。
本发明还提供一种所述的关键基因GhPIF7或所述的重组载体或所述的重组菌在改善陆地棉响应苗期高温胁迫的开花性状中的应用。
本发明还提供一种所述的关键基因GhPIF7或所述的重组载体或所述的重组菌在改善陆地棉响应高温胁迫的现蕾性状中的应用。
本发明还提供一种所述的关键基因GhPIF7或所述的重组载体或所述的重组菌在改善陆地棉响应高温胁迫的株高性状中的应用。
本发明公开了以下技术效果:
本发明采用转录组数据挖掘与环境温度相关的候选基因,发现GhPIF7基因可以响应高温胁迫,且在高温胁迫下该基因的表达量显著下降。为了进一步明确GhPIF7基因响应环境温度胁迫分子机理,将目的基因进行克隆,构建目的基因的VIGS沉默载体,研究在苗期相对高温胁迫下的生物学功能,结果发现,GhPIF7基因沉默导致陆地棉的株高变矮、现蕾和开花时间延迟,影响陆地棉的生长发育。以此证实GhPIF7基因正向调控棉花响应高温胁迫反应,改善高温对棉花的开花、现蕾、株高等表型生长发育的影响,为陆地棉耐高温胁迫育种提供重要基因资源。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为载体pGM-T和TRV载体的图谱;
图2为目的基因PCR扩增、菌液PCR和双酶切结果;A:GhPIF7基因的PCR扩增;B:GhPIF7的菌液PCR;C:双酶切检测阳性质粒;
图3为GhPIF7的克隆产物测序结果;
图4为目的基因的沉默及沉默效率的检测;A:TRV:PDS阳性对照表型;B:GhPIF7基因沉默效率检测;
图5为GhPIF7沉默植株的表型分析及高温胁迫对植株的现蕾时间和开花时间影响;
图6为GhPIF7基因的RNA-seq分析。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
本发明以陆地棉品种“中棉113”为研究对象,正常出苗后,分别在20℃(对照温度,Control temperature,CT)和30℃(适度高温,High temperature,HT)两个环境下放置棉苗7、14和21d三个不同时间段,上述处理分别命名为7、14、21CT和7、14、21HT。然后放置室温观察植株的形态特征,比较茎尖及幼叶在CT和HT两个环境下生理指标的变化趋势。对14CT、14HT、21CT、和21HT四个样本的茎尖进行RNA-seq,用其转录组数据挖掘与环境温度相关的候选基因,发现GhPIF7基因在温度处理14d和21d后其表达量呈相反趋势,且在30℃高温下与20℃温度下相比,GhPIF7基因的表达量显著下降(见图6),再通过qRT-PCR验证发现其表达水平与转录组水平一致。因此猜测这些基因有可能响应环境温度胁迫。为了进一步明确GhPIF7基因响应环境温度胁迫分子机理,将目的基因进行了克隆,构建目的基因的VIGS沉默载体,研究在苗期相对高温胁迫下的生物学功能。实验如下:
1试验材料与试剂
1.1试验材料
本试验所用材料为陆地棉中棉113(ZM113),种子是由中国农业科学院棉花研究所提供。
1.2试验试剂
本研究所用试剂见表1:
表1实验所用试剂
1.3菌株与载体
GV3101农杆菌感受态细胞和pGM-T克隆载体(VT302-02)分别购买自上海唯地生物技术有限公司和天根生化科技有限公司,用于基因沉默的VIGS载体系统(TRV156、TRV192和TRV-GhPDS)由中国农业科学院棉花研究所转基因课题组馈赠,pGM-T和TRV载体图谱如图1所示。
1.4溶液配制
a.培养基的配制见表2:
表2培养基配制
注:培养基需在121℃灭菌20min
b.100mL 50×TAE缓冲液:24.2g Tris+3.72gNa2EDTA·2H2O+5.71mL冰乙酸;
c.50mg/mL利福平(Rif)和20mg/mL乙酰丁香酮(AS):将5g Rif粉末和2g乙酰丁香酮粉末分别溶于100mL DMSO溶液中,分别用0.22的滤膜过滤除菌,封装,-20℃保存;
d.0.5M MES:将10.65g MES粉末溶于50mL ddH2O中,用NaOH将pH值调至5.6,之后定容至100mL,用0.22的滤膜过滤除菌,封装,4℃保存;
e.1M MgCl2:用100mL ddH2O溶9.521g MgCl2粉末(放大量热),0.22滤膜过滤除菌,4℃保存;
f.500mL重悬液:10mL 0.5M MES+1ml 20mg/mLAS+5mL 1M MgCl2,无菌ddH2O补足至500mL。
1.5使用仪器
主要仪器:可见分光光度计、高速冷冻离心机、水浴锅、电泳仪、人工气候箱、切胶仪、超低温冰箱、荧光定量仪、梯度PCR仪、灭菌锅、电子天平、微波炉、台式恒温摇床、制冰机、超净工作台、生化培养箱、超微量分光光度计、超纯水仪器和迷你漩涡混匀仪。
2试验方法
2.1引物设计
利用NCBI Primer-BLAST(https://www.ncbi.nlm.nih.gov/tools/primer-blast/)设计GhPIF7基因的克隆引物、qRT-PCR引物、VIGS沉默引物(VIGS沉默引物产物片段大小为300~500bp),引物序列如表3:
表3本实验所用的引物列表
2.2总DNA和RNA的提取
2.2.1DNA提取
a.在研钵中放入植物的顶端分生组织和液氮充分研磨至粉末,立即装入1.5mL离心管中;
b.向离心管中迅速加入400μL的GPS缓冲液和10μL的RNaseA,涡旋振荡,放在65℃水浴锅中15min;
c.水浴结束后,向离心管中加入100μLGPA缓冲液,振荡1min后,在12000rpm条件下离心5min;
d.转移上清液至过滤柱CS中,12000rpm离心1min,上清液转移至新的离心管;
e.加入等量的无水乙醇、混匀,转移至吸附柱CR2中,在12000rpm条件下,离心1min,弃废液;
f.将550μL去蛋白液RD加入CR2中,离心1min,倒掉废液;
g.将700μL漂洗液PW加入CR2中,离心1min,倒掉废液;
h.重复步骤g后,离心2min;
i.将CR2放入新的离心管,室温晾5-10min;
j.将70μL洗脱缓冲液TB加入CR2膜中间,放置3-5min,离心2min后,得到DNA溶液,分装,-20℃保存。
k.为了保证后续试验的准确性,将获得的DNA溶液用超微量分光光度计(MicroDrop)和1%的琼脂糖凝胶电泳检测其提取质量、含量和纯度。
2.2.2RNA提取和反转录
根据多糖多酚植物总RNA提取试剂盒说明书提取RNA。
反转录(cDNA第一链的合成):
a.将分装的RNA按照自己的需求从-80℃冰箱取出并在冰上融化,在-20℃冰箱中取出5×FastKing-RT SuperMix试剂和RNase-Free ddH2O在冰上融化,轻摇混匀;
b.反应体系:
表4反应体系
c.反应程序:
表5反应程序
d.反应完成后,检测cDNA的纯度及浓度,分装,-20℃保存。
2.3目的片段的扩增与克隆载体的连接和转化
2.3.1目的片段的扩增
以中棉113(ZM113)的cDNA为模板,利用2×Pro Taq预混液(含染料)试剂盒进行目的基因的扩增,体系如表6:
表6扩增体系
根据上述体系加完反应液后,轻摇混匀,短暂离心,按照表7反应程序进行反应:
表7反应程序
反应结束后,用1.8%的琼脂糖凝胶电泳检测其目的基因的大小是否合适。
采用PCR方法从陆地棉上扩增出GhPIF7基因全长,其核苷酸序列如SEQ ID NO:1所示。
克隆获得GhPIF7的基因序列(该序列包含5'UTR、3'UTR和CDS序列,SEQ ID NO:1):
GACAGTTTGAACTCAGAACTTCTAACTCCAAACTCAGCCTCAAAAGCTGCGGATTGAAACAAAACAAAAGAGAAAGAGAGTTTGAAAGAATCAAATCATCATGAGTCACTGTATAGTACCAAATTGGAATCTAAAAAACCAAAGGCGACGACGTCAACAAGTCGAAGAAGAAGAAGAGGAAACCAAGAGATCTTCGTTCCACATGTTCAACCCTTGTAATAATATAAACCAGAATCTCGTCCCCGTGTCCAACCATCAAGTTGCAGACCTAACATGGCAATATGGTCAGCAGCTAGCCATGCATGGGTTCAGTGGGTTCCTTCCTACTGCACCAACAAAGCCAACATGTAGGTCAAATGACACATTAGAGTCCATAGTCCATCAAGCCACATACCACAACCATGATGAGACCCCGACAAACACAAGTTCCATTGCTGCATCATCGGGGGGAAATCGCAGCGAAAGCTCCAGTCGATTGCCGCCGGTGTCGGTAGCAGCGCGGTTGACAAAGAAACGGGCACAGCCGATTTCAGATTCGGATCAATGCAGGAAACACAATATAAGTTGTGGGATTCAAAAAGATAAAGCAGATCGTAGTGAATGCGGCAGTGATACGTTTTACAAGATCGACAATGATGCAACAATGGTGACTTGGGGTTCCCATGATGAATCTCTTCAAAGCTTGAAAACCAAAACCACAGATGTGGACTCTGGTTGTCACGATGGATCGGAAAGCCGAGATGAAACAGGACGGTCTCATCCGACAAGGCGAAGTCGAGCAGCAGCTATTCATAATCTGTCTGAAAGGAAGCGAAGAGATAGAATTAATCAAAAGATGAAGGCTCTTCAAAAGCTGGTGCCAAATGCAAGCAAGACAGATAAAGCTTCAATGCTTGATGAAGTAATTGAATACCTAAAACAACTTCAAGCACAAGTACAAGTGATGAGTATGAGAAGCCTCCCTCCAATGATGTTGATGCCACTAGGCTTGCACCACCACCAGCATCTGCAAATGTCTCTTTTAGGTCGGATCATGGCCGGCATGGGAGTTAACCATGCTTTAGGAATGGGCATGGGATTGCTAGACATCAACGCCGCCACGCCTCCCAATGCTTCCCAATCTCTTCCTTCTCTATTGCACCTTCCACCACCCTTTTTGGCAACTGCACTGCCGCCTATGATTCCTTCAAGAGCTACCGCCACTGCAGCAGCTCAATCAAATCCAAATGCGAGCTCTAGTGACTCAATCCCTTTGCCTGACCCATCTTGCGCTTTCCTAACTCAATCCATGAATATGGAACTCTACAGTAAGATGGCAGCACTGTATCAGTCTCAAATGAATCGAACGACAGAGACAGCCAGCAGTCCATCGCGATCAAACAACATAAAACAGGATTGTTGTCGCATGATATAAGATCTTTGTATAATATAACTTAGATTTGTATGTCTTATAATTGCCTCCCCCCTTTTTTCTTTATATA TTTATCTGATTAGCC。
2.3.2目的片段的胶回收
a.将500μLBL平衡液加入CB2柱中(柱子放在收集管中),离心1min(转速和温度为12000rpm和4℃),倒掉收集管中的废液;
b.在切胶台上回收目的条带胶块(在切胶时紫外照射的时间不宜过长),放置在1.5mL离心管中进行称重,向离心管中加入PC试剂(每0.1g加100μL的PC试剂),在50℃的水浴锅中处理10min(期间上下翻转)后,放置室温待冷却后,加入已平衡好的CB2柱中,离心1min(条件与步骤a中一致,后续的离心条件都保持一致),倒掉废液;
c.在CB2中加入600μL加入了无水乙醇的PW溶液,室温放置2min,离心1min,倒掉废液;
d.重复步骤c的操作后,离心2min,室温放置5min,将CB2放入新的1.5mL离心管中,在吸附柱膜的中央(注意不要戳到膜)加入PH=7.0-8.5的ddH2O,放置2min后,离心2min,获得目的片段的DNA溶液,用超微量分光光度计(Micro Drop)检测其浓度和纯度后,分装,-20℃保存。
2.3.3目的片段与克隆载体pGM-T的连接与转化
a.将pGM-T载体从-80℃超低温冰箱中取出,在冰上融化;
b.根据表4-6向1.5mL离心管中加入反应液(整个操作在冰上完成):
表8连接体系
注:根据载体与目的片段的摩尔比=1:3-1:8计算得出目的片段的体积。
c.将所添加的组分轻柔混匀,短暂离心,16℃过夜。
2.3.4TOP 10大肠杆菌转化
a.平板制备:
参照1.4的方法,配制LB固体培养基,准备所需的培养皿,121℃灭菌20min;放置超净工作台中,等温度降至55℃左右时,往培养基中加入IPTG、X-Gal和Amp+使其终浓度为100μg/mL、125μg/mL和100μg/mL,充分混匀后,倒入灭了菌的培养皿中,在37℃倒置3h,备用;
b.转化
1.从-80℃超低温冰箱取出TOP10感受态细胞,在冰上融化,将一管分装成两管,加入5μl连接产物,轻弹混匀,按照表4-7顺序进行反应(整个操作在冰上完成):
表9反应时间
2.冰浴3min结束后,加入不加抗生素LB液体培养基250μL(37℃预热),振荡培养45min(37℃,150rpm);
3.培养完成后,吸取100μL溶液(稀释200倍),使用涂布棒均匀的涂抹在固体培养基中、封口,培养12h(37℃,黑暗);
4.12h后,平板上会出现蓝色和白色的菌斑,将白色单菌落用牙签挑至已加好抗生素Amp+的5mL LB液体培养基中,在37℃,振荡培养12-16h(200rpm);
5.通过菌液PCR检测阳性,并提取质粒。
2.3.5质粒的提取
a.将500μL平衡液BL加入吸附柱CP3中,12000rpm条件下,离心1min,弃废液;
b.将5mL菌液在12000mL条件下,离心1min,收集菌体,尽量吸除上清;
c.加入250μL的溶液P1(已加入RNase A),用移液器吹打使细菌彻底悬浮;
d.加入250μL溶液P2,迅速温和地翻转7次,持续时间不能超过5min;
e.加入350μL溶液P3,迅速温和地翻转7次,离心10min;
f.转移上清液至CP3中,离心45sec,弃废液;
g.加入600μL漂洗液PW,12000rpm离心45sec,倒掉废液;
h.重复步骤g后,12000rpm离心2min;
i.将60μLPH=7.5的ddH2O加入CP3膜中间,放置2min,离心2min,得到质粒溶液,分装,-20℃保存。
j.以质粒为模板做PCR检测阳性,并将其送公司测序(上海生工)。
2.4沉默载体的构建与转化
2.4.1沉默载体构建
以2.3.5中测序成功的阳性质粒为模板,用加了限制性酶的酶切位点(EcoR I和Xho I),并且添加了保护碱基的引物进行PCR扩增,通过将PCR产物和TRV-156载体进行双酶切的方法将GhPIF7、GhCDF3和GhZAT10基因的片段插入到TRV-156载体中,形成TRV:GhPIF7、TRV:GhCDF3和TRV:ZAT10构建体,酶切体系如表10:
表10酶切体系
体系完成后37℃反应3h,待结束加10×Loading Buffer从而停止反应;胶回收酶切产物。之后将目的片段与沉默载体连接;转化到大肠杆菌进行扩繁,37℃培养12h,挑菌,振荡培养,之后进行菌液PCR和质粒双酶切鉴定,完成后将阳性质粒送至上海生工测序。
2.4.2转化农杆菌GV3101
a.将GV3101感受态细胞从-80℃超低温冰箱取出,在冰上解冻,分装成两管,吸取2μL测序成功的质粒,加入离心管中,按表11顺序进行转化:
表11反应时间
b.完成上述步骤后,加入350μL的LB液体培养基(未加抗生素),振荡培养2h(28℃,200rpm),将菌液均匀的涂布在固体培养基中(添加Kan+和Rif抗生素),在黑暗、28℃条件下,培养2天。
c.培养完成后,将单菌落挑至5mL LB液体培养基中(添加Kan+和Rif抗生素),按照步骤b中振荡培养的条件进行培养12-16h;
d.培养完成后,用50%甘油保存菌液(菌液:甘油=1:1),-80℃保存备用;做菌液PCR确认载体阳性。
2.4.3陆地棉植株VIGS沉默
a.在每个培养钵中装入相同重量的营养土(基质:蛭石=1:1),将其浸水直至表面湿润,用于种子萌发。选取健康有活力的ZM113种子,置于10×15cm的培养钵中,种植的深度一致,在人工气候箱温室培养,待其生长至第7天,子叶完全展开时,将其浸水,待营养钵中的土达到表面湿润后,停止浸水,放置备用;
b.将从-80℃取出的VIGS载体系统和目的基因沉默载体的菌液在冰上融化,按照菌液:LB液体培养基=1:10的量将菌液进行活化,活化完成后,按照同样比例进行扩繁;
c.菌液扩繁完后,在5000rpm的条件下,离心10min,保留菌体,倒掉上清液,用MMA使菌体悬浮,并在600波长下测定OD值使其在1.8左右;
d.重悬后,黑暗放置3-5h,将TRV192分别与含TRV156(为TRV:156,空白组)、TRV:GhPDS(为阳性对照)、TRV156:GhPIF7(实验组)、TRV156:GhCDF3和TRV156:GhZAT10的菌体重悬液1:1混合,并将其充分混匀,然后在棉花子叶背面用1mL注射器注射菌体,使菌液充满整个叶片;
e.将注射的苗子放置25℃黑暗条件下放置24h后,在正常生长条件下培养。
2.5沉默植株的鉴定
待阳性对照棉花幼苗出现白化后,采取实验组及空白组的棉花幼叶进行荧光定量实验,检测其沉默效率。
2.6沉默植株的高温胁迫处理
待注射过的阳性棉花幼苗出现白化后,将空白组和实验组生长至四周龄的棉花幼苗分别置于20℃和30℃环境7d,进行适当高温胁迫处理,进行表型观察。
3结果与分析
3.1沉默载体的构建与沉默株系的阳性鉴定
3.1.1目的基因片段的扩增与沉默载体的构建
用NCBI的blast-primer设计引物,以中棉113的cDNA为模板,使用艾科瑞的2×ProTaq酶进行目的片段的扩增,使用通用型DNA纯化回收试剂盒(天根)回收目的片段(图2中A)。将回收的目的片段与pGM-T载体连接进行蓝白斑筛选,提质粒。通过双酶切的方法将目的片段与TRV156载体连接并培养,通过菌液PCR(图2中B)、双酶切(图2中C)和测序(图3)方法得到阳性质粒。根据测序可得连接到该载体上的目的片段并未发生碱基的缺失和替换,因此测序成功。将阳性质粒用冻融法转化到农杆菌进行培养。
3.1.2目的基因的沉默效率检测及表型分析
基于转录组数据分析,筛选出温度影响陆地棉生长发育的候选基因。因此利用VIGS技术研究了温度胁迫下目的基因在陆地棉中的作用。我们选择基因GhPIF7进行VIGS试验。通过在子叶下表皮注射的方法,沉默陆地棉中棉113中的目的基因,发现在感染后的第7天,阳性对照(TRV:GhPDS)植株开始白化,沉默后的第12天,阳性对照白化明显(图4中A),说明农杆菌感染陆地棉成功。对目的基因的沉默效率检测发现(图4中B),与对照组相比,TRV:GhPIF7植株中GhPIF7的表达明显受到抑制,GhPIF7基因的沉默效率为60.61%。通过VIGS沉默植株和对照植株的表达量分析,说明目的基因已被沉默。
3.1.3适当高温胁迫对沉默植株的表型影响
挑选四周龄的TRV:GhPDS、TRV:156、TRV:GhPIF7植株分别置于20℃和30℃环境7d,进行适当高温胁迫处理。之后观察其株高、现蕾和开花时间(图5),发现将GhPIF7基因沉默之后,陆地棉的株高变矮、现蕾和开花时间延迟。同时我们将GhPIF7基因沉默株系进行温度处理后,发现响应适当高温的调控,相较于空白对照组植株,沉默植株在高温胁迫下的现蕾时间和开花时间均延迟,影响棉花生长发育。发现将该基因沉默之后,陆地棉GhPIF7沉默植株的株高降低了3.59cm、平均现蕾和开花时间分别延迟了14.3天和11.6天。同时将GhPIF7基因沉默株系进行30℃和20℃温度处理后,发现陆地棉GhPIF7沉默株系在30℃处理7天后现蕾和开花时间比对照延迟9.5天和14.17天,均达到显著水平;而在20℃处理7天时,其现蕾和开花时间与对照无显著性差异。上述结果表明GhPIF7基因响应环境温度调控陆地棉开花时间。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (4)
1.一个响应环境温度调控陆地棉开花的关键基因GhPIF7,其特征在于,其核苷酸序列如SEQ ID NO:1所示。
2.一种如权利要求1所述的关键基因GhPIF7在延迟陆地棉响应高温胁迫的开花时间中的应用,其特征在于,通过在所述陆地棉中沉默所述关键基因GhPIF7,以延迟陆地棉响应高温胁迫的开花时间。
3.一种如权利要求1所述的关键基因GhPIF7在延迟陆地棉响应高温胁迫的现蕾时间中的应用,其特征在于,通过在所述陆地棉中沉默所述关键基因GhPIF7,以延迟陆地棉响应高温胁迫的现蕾时间。
4.一种如权利要求1所述的关键基因GhPIF7在降低陆地棉响应高温胁迫的株高性状中的应用,其特征在于,通过在所述陆地棉中沉默所述关键基因GhPIF7,以降低陆地棉响应高温胁迫的株高性状。
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