CN114807172B - 一种盐响应的玉米功能基因 - Google Patents
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Abstract
本发明公开了一种盐响应的玉米功能基因。所述功能基因为ZmBSN3,其核苷酸序列如序列表SEQ ID NO:1所示,其表达的蛋白序列如序列表SEQ IDNO:2所示。本发明以耐盐与盐敏感自交系构建F2群体,以F2群体为研究对象,进行BSA分析,获得一个耐盐基因ZmBSN3,该耐盐基因与K+、Ca2+的积累高度相关。为选育高耐盐玉米品种打下了良好的理论基础。
Description
技术领域
本发明属于分子育种生物学技术领域,具体涉及一种盐响应的玉米功能基因及其应用。
背景技术
玉米(Zea mays L.)是禾本科的一年生草本植物,是世界重要的粮食作物。玉米中的维生素含量非常高,是稻米、小麦的5-10倍,在所有主食中,玉米的营养价值和保健作用是最高的。玉米中含有的核黄素等高营养物质,对人体是十分有益的。
盐胁迫是影响玉米生长和产量的一个重要环境限制因素,盐胁迫易引发离子胁迫和渗透胁迫,最终导致植物叶面积扩展受阻、光合作用以及生物量积累降低等。我国有大面积的盐碱地,玉米在这些盐碱地上生长,产量很低。从基因改造的角度改善玉米的耐盐特性,研究的最为广泛,已报道的耐盐相关基因有:ZmNAC89、PPase、CIPK42、ZmKNOLLE等。开发新的耐盐基因,从而为耐盐玉米品种的选育打下基础,仍然具有重大意义。
发明内容
本发明的目的在于提供盐响应的玉米功能基因及其应用。
本发明以耐盐(NX388)与盐敏感(NX420)自交系构建F2群体,以F2群体为研究对象,进行BSA分析,结果获得一个候选基因ZmBSN3。
一种盐响应的玉米功能基因,所述功能基因为ZmBSN3,其核苷酸序列如序列表SEQID NO:1所示。
所述功能基因表达的蛋白序列如序列表SEQ ID NO:2所示。
一种含所述盐响应的玉米功能基因的载体。
含有所述盐响应的玉米功能基因载体的重组菌或细胞系。
扩增所述的盐响应的玉米功能基因中任一片段的引物。
所述盐响应的玉米功能基因在提高植物耐盐方面的应用。
所述植物为玉米、小麦、大豆、高粱、油菜或棉花。
本发明的有益效果:本发明以耐盐(NX388)与盐敏感(NX420)自交系构建F2群体,以F2群体为研究对象,进行BSA分析,获得一个耐盐基因ZmBSN3,该耐盐基因与K+、Ca2+的积累高度相关。为选育高耐盐玉米品种打下了良好的理论基础。
附图说明
图1为耐盐(NX388)与盐敏感(NX420)植株在正常培养和盐胁迫下的表型。
图2为定位基因ZmBSN3的qRT-PCR分析。
图3为载体连接方案。
图4为载体结构示意图。
图5为基因编辑突变体ZmBSN3Crispr表型分析。
图6为基因编辑突变体ZmBSN3Crispr离子分析。
具体实施方式
为了便于理解本发明,下面将对本发明进行更全面的描述。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
实施例1
以耐盐(NX388)与盐敏感(NX420)自交系构建F2群体,以F2群体为研究对象(图1),进行BSA分析,结果获得一个候选基因ZmBSN3。
所述耐盐基因被命名为ZmBSN3,其核苷酸序列如序列表SEQ ID NO:1所示。所述耐盐基因表达的蛋白序列如序列表SEQ ID NO:2所示。
实施例2定位基因ZmBSN3(Zm00001d0680)的表达分析
对亲本材料在150mM NaCl胁迫14天后取其根、叶,提取RNA,进行qRT-PCR,结果显示,在高盐胁迫下,在胁迫48H后,抗性亲本NX388叶片中ZmBSN3表达量明显高于敏感亲本,结果见图2。
实施例3基因编辑突变体表型分析
构建玉米基因Zm00001d040680敲除载体986,对ZmBSN3基因进行crispr敲除,设计三个靶点,构建编辑载体,构建过程如下:
一、预选靶标
T1:TATTCAATCAGGCATCTGCAGGG;
T2:TCAATATCCGGTTCAAGTGCAGG;
T3:TGATCCCATATCTGGTGAAGAGG。
二、构建中间载体
1、引物合成
986-T1引物:
986-T1+:cagtggtctcaggcaTATTCAATCAGGCATCTGCA;
986-T1-:cagtggtctcaaaacTGCAGATGCCTGATTGAATA;
986-T2引物:
986-T2+:cagtggtctcagccgTCAATATCCGGTTCAAGTGC;
986-T2-:cagtggtctcaaaacGCACTTGAACCGGATATTGA;
986-T3引物:
986-T3+:cagtggtctcaatgtTGATCCCATATCTGGTGAAG;
986-T3-:cagtggtctcaaaacCTTCACCAGATATGGGATCA。
2、引物在55℃变性、14℃退火,得到gRNA片段。
3、酶切连接
将上述gRNA片段分别用ECO31I酶切,通过T4链接试剂盒分别将三个gRNA连入空载体,具体反应体系:空载体1.5μL、gRNA片段2μL、ECO31I 0.5μL、T4-ligase 0.5μL、T4-buffer 1μL、H20 4.5μL,把配置好的体系置于37℃培养箱约2h。
4、重组质粒转化:将上述重组质粒利用热激法转化DH5感受态,37℃过夜培养。
5、阳性克隆检测引物
F链:
空载1:sgA-T:GACCATAGCACAAGACAGGCGT;
空载2:sgB-T:CGAATGAGCCCTGAAGTCTGAAC;
空载3:sgC-T:CATTTCATTACCTCTTTCTCC;
R链:靶位点引物的R链
挑选正确的单克隆,摇菌。
6、抽提质粒
具体操作步骤如下:
利用天根质利提取试剂盒进行质利提取,取微量回收产物,采用浓度为1%的琼脂糖凝胶电泳检测质粒抽提质量。
7、测序:把抽提的质粒各送2个单克隆,测序。
8、终载体连接
采用同尾酶连接的方法,一次将多个含有靶位点的中间载体片段连入终载体,载体连接方案如图3所示,载体结构如图4所示。
连接体系:
9、终载体检测引物
pOSCas9-S:GATGGGTTTTTATGATTAGAGTCC;
强cas9-:ggctcgtatgttgtgtgg;
大小:1600bp左右。
采用常规方法转化,获得该基因的突变体ZmBSN3Crispr,并进行了盐胁迫试验,未经胁迫处理的WT、ZmBSN3Crispr叶片中K+没有明显的差异,而在150mM NaCl处理14天后,ZmBSN3Crispr出现明显受阻,且叶片出现明显的干枯(图5),同时,叶片中K+、Ca2+显著高于其WT,说明ZmBSN3与K+、Ca2+的积累高度相关(图6)。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 宁夏农林科学院农业生物技术研究中心(宁夏农业生物技术重点实验室)
中国农业科学院生物技术研究所
<120> 一种盐响应的玉米功能基因
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 345
<212> DNA
<213> 玉米(Zea mays L.)
<400> 1
atgtcacgca aactaaaaat tttcagtgat cagatcaaca gggcaggttt aagatcttct 60
gttcgacctg cacttgaacc ggatattgat ttggaggagc tagagggagt gcatcaagga 120
gcacatggac attctggagt gagatttgtt agtggaataa tattaaagtc gaaggcactt 180
gcttttgagc ggatgctttt tcgagctaca agaggaaaca tgttattcaa tcaggcatct 240
gcaggggaac ctgtaactga tcccatatct ggtgaagagg tagagaaagc tgtttttgta 300
gttttcttct ggggaacaag caaaagcaaa gatactaaaa actag 345
<210> 2
<211> 114
<212> PRT
<213> 玉米(Zea mays L.)
<400> 2
Met Ser Arg Lys Leu Lys Ile Phe Ser Asp Gln Ile Asn Arg Ala Gly
1 5 10 15
Leu Arg Ser Ser Val Arg Pro Ala Leu Glu Pro Asp Ile Asp Leu Glu
20 25 30
Glu Leu Glu Gly Val His Gln Gly Ala His Gly His Ser Gly Val Arg
35 40 45
Phe Val Ser Gly Ile Ile Leu Lys Ser Lys Ala Leu Ala Phe Glu Arg
50 55 60
Met Leu Phe Arg Ala Thr Arg Gly Asn Met Leu Phe Asn Gln Ala Ser
65 70 75 80
Ala Gly Glu Pro Val Thr Asp Pro Ile Ser Gly Glu Glu Val Glu Lys
85 90 95
Ala Val Phe Val Val Phe Phe Trp Gly Thr Ser Lys Ser Lys Asp Thr
100 105 110
Lys Asn
Claims (1)
1.敲除ZmBSN3基因在降低玉米耐盐方面的应用,其特征在于,所述ZmBSN3基因的核苷酸序列如序列表SEQ ID NO:1所示。
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---|
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毛毅辉 ; 王天宇 ; 荣廷昭 ; 石云素 ; 宋燕春 ; 黎裕 ; .玉米V-ATPase B亚基基因(ZmVHA-B)的克隆及其表达分析.玉米科学.2009,(第02期),摘要. * |
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