CN114292857A - 一种花生耐盐基因AhMADS50及其应用 - Google Patents
一种花生耐盐基因AhMADS50及其应用 Download PDFInfo
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- CN114292857A CN114292857A CN202111597496.1A CN202111597496A CN114292857A CN 114292857 A CN114292857 A CN 114292857A CN 202111597496 A CN202111597496 A CN 202111597496A CN 114292857 A CN114292857 A CN 114292857A
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- ahmads50
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- peanut
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Abstract
本发明公开了一种花生耐盐基因AhMADS50及其应用。本发明筛选并克隆一个花生耐盐基因AhMADS50,其核苷酸序列如SEQ ID No.1所示,编码的氨基酸序列如SEQ ID No.2所示。本发明还公开了花生耐盐基因AhMADS50的扩增引物。所述花生耐盐基因AhMADS50经克隆、构建超表达载体、过表达重组菌株后,转化到拟南芥中获得过表达转基因拟南芥植株,其耐盐性得到明显提升。因此,花生耐盐基因AhMADS50的鉴定和耐盐功能分析丰富了花生耐盐育种的基因资源,对耐盐植物的育种和筛选具有重要的理论意义和应用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及一种花生耐盐基因AhMADS50及其应用。
背景技术
花生(Arachis hypogaea L.)是我国重要的油料作物,是优质植物油脂和蛋白质的核心来源之一。随着人类对花生的需求量持续增加,而耕地面积减少和粮食生产压力增加,粮油作物争地矛盾愈加突出,严重限制花生种植规模和供应。我国盐碱地面积9900万hm2以上,占全国耕地面积的6.62%,具有重要的农业开发潜力和前景。研究植物耐盐碱生理和信号机制,选育耐盐碱作物品种,是高效利用盐碱地这一战略后备土地资源的有效途径。
研究表明,花生对盐胁迫具有一定耐受性,培育高耐盐性花生新品种,是扩大种植面积和提高产量的有效手段。因此,鉴定耐盐基因,研究其在花生耐盐调控中的生理功能和分子机制,构建综合调控网络,并集成转基因和分子辅助育种技术选育耐盐碱花生新品种,对保障花生抗逆高产和花生产业可持续发展意义重大,具有强烈地必要性和迫切性。
发明内容
本发明提供了一种花生耐盐基因AhMADS50及其应用。本发明从耐盐花生品种中筛选分离和克隆到AhMADS50基因,并证实了该基因可以有效提高拟南芥的耐盐性。
为实现上述发明目的,本发明采用以下技术方案予以实现:
本发明提供了一种花生耐盐基因AhMADS50,所述花生耐盐基因AhMADS50的核苷酸序列如SEQ ID No.1所示,其编码的氨基酸序列如SEQ ID No.2所示。
本发明还提供了扩增所述的花生耐盐基因AhMADS50的引物,所述引物的核苷酸序列为:
AhMADS50-F:ATGGGTCGTGGAAAGATTG;
AhMADS50-R:TTAAGCGAGGCGGAGATC。
本发明还提供了所述的花生耐盐基因AhMADS50在提高植物耐盐性中的应用。
进一步的,所述应用包括以下步骤:
(1)PCR扩增及克隆AhMADS50基因;
(2)克隆的AhMADS50基因全长CDS连入表达载体中,得到过表达重组载体;
(3)将过表达重组载体转化到农杆菌中,得到过表达重组菌株;
(4)将过表达重组菌株转化进植物中,筛选并得到AhMADS50基因过表达株系。
进一步的,所述步骤(1)中PCR的扩增条件为:98 ℃ 1 min;98 ℃ 10 s,58℃ 15s,68 ℃ 40 s,重复30循环;68 ℃ 3 min。
进一步的,所述步骤(2)中表达载体为pCambia2300,该载体启动子为35S,筛选标记为抗除草剂基因BAR。
进一步的,所述AhMADS50基因过表达株系与野生型株系相比,耐盐性得到明显提升。
进一步的,所述AhMADS50基因过表达株系与野生型株系相比,在盐胁迫条件下,植物种子发芽率明显增加。
进一步的,所述植物为拟南芥。
本发明还提供了所述的花生耐盐基因AhMADS50或所述的花生耐盐基因AhMADS50的引物在用于选育耐盐花生品种中的应用。
本发明与现有技术相比,具有以下优点和有益效果:
本发明从耐盐花生品种“花育22号”中筛选并克隆了一个耐盐基因AhMADS50,通过荧光定量 PCR 验证了AhMADS50在盐胁迫下的表达模式,该基因盐胁迫下在叶片和根部的转录水平均有显著提高。本发明还利用该基因经克隆、构建超表达载体、过表达重组菌株后,转化到拟南芥中获得过表达转基因拟南芥植株 AhM50-OX,且经实验验证AhM50-OX在盐胁迫下的发芽率明显高于野生型。因此,花生AhMADS50基因的鉴定和耐盐功能分析丰富了花生耐盐育种的基因资源,对耐盐植物的育种和筛选具有重要的理论意义和应用价值。
附图说明
图1为盐胁迫下AhMADS50基因在叶片和根中的表达模式。
图2为转基因植株AhM50-OX耐盐性鉴定。
具体实施方式
结合以下具体实例对本发明的技术方案作进一步详细的说明。
下述实施例中,如无特殊说明,所使用的实验方法均为常规方法,所用材料、试剂等均可从生物或化学试剂公司购买。
实施例1:AhMADS50基因的克隆
本发明筛选并克隆了一个耐盐基因并命名为AhMADS50,具体步骤为:
提取耐盐花生品种“花育22号”幼嫩叶片RNA,将其反转录成cDNA,以此为模板,使用引物扩增AhMADS50基因,将扩增序列进行测序,确定目的基因的核苷酸序列。
扩增引物为:
AhMADS50-F:ATGGGTCGTGGAAAGATTG(SEQ ID No.3);
AhMADS50-R:TTAAGCGAGGCGGAGATC(SEQ ID No.4)。
扩增花生AhMADS50基因的PCR扩增体系为:5 μL 5× PrimeSTAR GXL Buffer、1 μL dNTP Mixture、1μL总 cDNA、1 μL AhMADS50-F、1 μL AhMADS50-R、1 μL PrimeSTAR GXLDNA Polymerase和9 μL无菌双蒸水。
扩增花生AhMADS50基因的PCR反应条件为:98 ℃ 1 min;98 ℃ 10 s,58℃ 15 s,68 ℃ 40 s,重复30循环;68 ℃ 3 min。
测序结果显示,AhMADS50基因的核苷酸序列如SEQ ID No.1所示,其编码的氨基酸序列如SEQ ID No.2所示。
然后采用无缝克隆技术,将克隆到的AhMADS50基因全长CDS连入表达载体pCambia2300,该载体启动子为35S,筛选标记为抗除草剂基因BAR,得到过表达载体pCambia- AhMADS50。将过表达载体转化到农杆菌,然后利用花絮侵染法转化拟南芥,获得转基因植株AhM50-OX。
实施例2:荧光定量PCR对AhMADS50基因在盐胁迫下的表达模式进行分析
用100 mM 的 NaCl 溶液处理水培约 15 天的花生幼苗(花育22号),利用荧光定量PCR技术分析AhMADS50基因在0h、1h、6h、24h和48h盐胁迫处理后叶片和根组织中相对表达量变化。
其中,荧光定量PCR的引物序列为:
RTAhMADS50-F: GAAGACAGATCAGGAATCGGAT(SEQ ID No.5);
RTAhMADS50-R: CTCCAAGCTTCTCACCTTTTTC(SEQ ID No.6)。
同时以花生Actin作为内参基因,扩增的正向引物序列为:Actin-F:5’-TTGGAATGGGTCAGAAGGATGC-3’(SEQ ID No.7);反向引物序列为:Actin-R:5’-AGTGGTGCCTCAGTAAGAAGC-3’(SEQ ID No.8)。
结果如图1所示,AhMADS50基因在盐胁迫后的叶片和根中相对表达量均明显上升,1h后表达量开始有明显上升,6h达到峰值,由此验证了该基因对盐胁迫响应的敏感性。
实施例3:转基因拟南芥耐盐性鉴定
分别配制 NaCl 浓度为 0 mM和 150 mM 的 1/2 MS 培养基。收集实施例1中农杆菌侵染后的转基因拟南芥AhM50-OX的T0代种子,经消毒处理后,分散于上述配制的1/2 MS培养基的一半上,另一半分散放置等量的野生型拟南芥种子。观察、统计转基因和野生型拟南芥植株在发芽率这一耐盐生理指标的变化。
结果如图2所示,在含有150mM NaCl培养基上,AhM50-OX发芽率明显高于野生型拟南芥,表明转基因拟南芥的耐盐性优于野生型。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
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Claims (10)
1.一种花生耐盐基因AhMADS50,其特征在于,所述花生耐盐基因AhMADS50的核苷酸序列如SEQ ID No.1所示,其编码的氨基酸序列如SEQ ID No.2所示。
2.扩增权利要求1所述的花生耐盐基因AhMADS50的引物,其特征在于,所述引物的核苷酸序列为:
AhMADS50-F:ATGGGTCGTGGAAAGATTG;
AhMADS50-R:TTAAGCGAGGCGGAGATC。
3.权利要求1所述的花生耐盐基因AhMADS50在提高植物耐盐性中的应用。
4.根据权利要求3所述的花生耐盐基因AhMADS50在提高植物耐盐性中的应用,其特征在于,所述应用包括以下步骤:
(1)PCR扩增及克隆AhMADS50基因;
(2)克隆的AhMADS50基因全长CDS连入表达载体中,得到过表达重组载体;
(3)将过表达重组载体转化到农杆菌中,得到过表达重组菌株;
(4)将过表达重组菌株转化进植物中,筛选并得到AhMADS50基因过表达株系。
5.根据权利要求4所述的花生耐盐基因AhMADS50在提高植物耐盐性中的应用,其特征在于,所述步骤(1)中PCR的扩增条件为:98 ℃ 1 min;98 ℃ 10 s,58℃ 15 s,68 ℃ 40s,重复30循环;68 ℃ 3 min。
6.根据权利要求4所述的花生耐盐基因AhMADS50在提高植物耐盐性中的应用,其特征在于,所述步骤(2)中表达载体为pCambia2300,该载体启动子为35S,筛选标记为抗除草剂基因BAR。
7.根据权利要求4所述的花生耐盐基因AhMADS50在提高植物耐盐性中的应用,其特征在于,所述AhMADS50基因过表达株系与野生型株系相比,耐盐性得到明显提升。
8.根据权利要求4所述的花生耐盐基因AhMADS50在提高植物耐盐性中的应用,其特征在于,所述AhMADS50基因过表达株系与野生型株系相比,在盐胁迫条件下,植物种子发芽率明显增加。
9.根据权利要求3所述的花生耐盐基因AhMADS50在提高植物耐盐性中的应用,其特征在于,所述植物为拟南芥。
10.权利要求1所述的花生耐盐基因AhMADS50或权利要求2所述的花生耐盐基因AhMADS50的引物在用于选育耐盐花生品种中的应用。
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