CN115094073B - GmSKP1基因在负调控大豆干旱胁迫应答中的应用 - Google Patents
GmSKP1基因在负调控大豆干旱胁迫应答中的应用 Download PDFInfo
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Abstract
本发明提供了一种GmSKP1基因在负调控大豆干旱胁迫应答中的应用，属于基因调控技术领域。本发明发现GmSKP1基因可作为一个负调控因子参与大豆对干旱胁迫的响应，通过过表达株系的表型鉴定发现，其有效减少了大豆对干旱的耐受性；该发现的获得对于研究大豆干旱胁迫应答分子机理研究，通过基因编辑方法培育耐旱大豆新品种拓展了有效解决途径。
Description
技术领域
本发明属于基因调控技术领域，尤其涉及一种GmSKP1基因在负调控大豆干旱胁迫应答中的应用。
背景技术
植物会遭受各种非生物胁迫，例如：干旱、高盐度和冷冻等，这些胁迫会干扰细胞的水平衡，影响植物的生长，从而影响作物的生产力。并且非生物胁迫可导致植物一系列的生理和生化反应，例如：促进气孔关闭、影响细胞的分裂和伸长、改变细胞壁的弹性、抑制光合作用以及激活呼吸作用等，从而影响植物的生长和发育。
大豆作为全球最重要的农作物之一，其种子中含有丰富的蛋白和油，被用于人类食品或者动物饲料。干旱、高盐和低温等一系列非生物胁迫因素对大豆的生长、发育以及生产量造成严重威胁。在大豆植株的各组分中，根具有非常好的可塑性，但是至今为止对于调控根发育的可塑性分子机制仍然存在许多的未知。大豆根是土壤中第一个被逆境胁迫破坏的植物器官，对于研究大豆的抗逆性很关键。然而要确保在逆境中大豆高产机制的成功建立，大豆根抗逆的遗传机制有待探索。因此，探寻大豆抗旱的新机制对于加快大豆抗旱品种育种进程具有重要意义。
发明内容
本发明提供了一种GmSKP1基因在负调控大豆干旱胁迫应答中的应用，该应用的获得对于研究大豆干旱胁迫应答分子机理研究，通过基因编辑方法培育耐旱大豆新品种拓展了有效解决途径。
为了达到上述目的，本发明提供了一种GmSKP1基因在负调控大豆干旱胁迫应答中的应用。
作为优选，GmSKP1基因通过脯氨酸和可溶性糖含量来影响大豆对干旱胁迫的耐受性。
作为优选，所述干旱胁迫的条件为10％PEG的水中培养4-6天。
作为优选，在干旱迫条件下，相比较对照植株，过表达GmSKP1基因的复合体大豆中可溶性糖和脯氨酸含量均降低，从而降低其耐盐性。
作为优选，所述GmSKP1基因的核苷酸序列如SEQ ID NO：1所示。
作为优选，所述GmSKP1基因的氨基酸序列如SEQ ID NO：2所示。
作为优选，通过对GmSKP1基因的编码区进行基因编辑或RNAi，致使GmSKP1基因功能丧失，从而产生抗旱大豆植物。
本发明提供了一种提高大豆干旱胁迫应答能力的方法，包括以下步骤：
构建大豆基因GmSKP1的RNAi载体，经发根农杆菌介导转化到大豆中，获得提高大豆干旱胁迫应答能力的转基因植株。
作为优选，构建大豆基因GmSKP1的RNAi载体的方法为：
根据GmSKP1序列设计以下引物：
GmSKP1-RNAi-F：
5'-CATGCATCAAGAGCCTGCTGGACCTTATTCAAGAGATAAGGTCCAGCAGGCTCTTGATG-3'
GmSKP1-RNAi-R：
5'-CATGCATCAAGAGCCTGCTGGACCTTATCTCTTGAATAAGGTCCAGCAGGCTCTTGATG-3'
具体方法如下：
将上述引物退火后形成带有酶切位点的双链，并插入pCAMBIA3301载体，构建GmSKP1-RNAi-pCAMBIA3301重组质粒；
将上述GmSKP1-RNAi-pCAMBIA3301重组质粒转化发根农杆菌K599，得到RNAi抑制载体。
作为优选，构建大豆基因GmSKP1的RNAi抑制载体后，利用发根农杆菌进行大豆转化产生转基因毛状根，通过对所得转基因植株的GmSKP1基因表达量分析，得到提高大豆干旱胁迫应答能力的转基因植株。
与现有技术相比，本发明的优点和积极效果在于：
本发明发现GmSKP1基因可作为一个负调控因子参与大豆对干旱胁迫的响应，通过过表达株系的表型鉴定发现，其有效降低了大豆对干旱的耐受性；通过生理数据进一步发现，GmSKP1基因可能通过影响脯氨酸和可溶性糖含量来调节大豆对干旱胁迫的响应。该发现的获得对于研究大豆干旱胁迫应答分子机理研究，通过基因编辑方法培育耐旱大豆新品种拓展了有效解决途径。
附图说明
图1为GmSKP1-pEGAD转化K599农杆菌PCR检测示意图，其中M:DL2000；1-5:GmSKP1-pEGAD-k599；
图2为GmSKP1-pEGAD-k599毛状根DNA PCR检测示意图，其中M:DL2000；1-3:GmSKP1-pEGAD-K599；
图3为野生型和转基大豆qRT-PCR示意图；
图4为转基因毛状根大豆复合体植株PEG胁迫示意图；
图5为对照大豆和转GmSKP1大豆的存活率示意图；
图6为转基因毛状根大豆复合体植株生理指标测定示意图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1植物表达载体的构建
为分析GmSKP1在干旱胁迫应答的功能，根据GmSKP1的基因序列设计引物，以大豆叶片基因组作为DNA模板，扩增得到大豆GmSKP1基因(核苷酸序列如SEQ ID NO：1所示，氨基酸序列如SEQ ID NO：2所示)，并插入pEGAD载体，构建GmSKP1-pEGAD重组质粒。
1.1重组质粒GmSKP1-pEGAD转化发根农杆菌K599
具体操作步骤如下：
(1)挑一个单克隆的K599农杆菌加入到带链霉素抗性的1ml LB培养基中，在恒温摇床中28℃振荡过夜培养；
(2)移液枪吸取0.1ml将过夜培养菌液转移到20ml LB液体培养基中，另外加入20μl 50mg/L的链霉素，在恒温摇床中28℃振荡培养至OD值为0.8；
(3)低温离心机离心菌液，4000rpm离心5min，倒去上层清液；
(4)加入30％甘油氯化钙(CaCl2)重悬菌体；
(5)将感受态菌液用移液枪分装到1.5ml离心管中，每支离心管分装200μl感受态细胞，先经过冰水浴处理后，转移至液氮速冻，置于-80℃保存备用。
(6)向200μl冰浴解冻后的装有K599感受态细胞的离心管中加入10μl过表达载体质粒，轻轻用移液枪转动，使质粒与感受态细胞充分接触，在冰浴30min，上面过程均需在紫外灭菌过后的超净工作台中进行；
(7)液氮速冻离心管7min，迅速将离心管转移到37℃的水浴锅中孵育8min；
(8)加入600μl的LB液体培养基，28℃振荡培养3-4h；
(9)短暂离心后弃去部分上清液，混匀后吸取0.15ml涂布于双抗(链霉素和卡那霉素)的LB固体培养基平板上；28℃倒置培养2天可见菌落长出；
(10)对单克隆菌株进行PCR鉴定，确认菌液中含有构建正确的表达载体。
采用PCR方法从大豆叶片基因组DNA中扩增GmSKP1，获得了大小为468bp的片段，将GmSKP1插入到pEGAD载体上，采用载体通用引物经菌液PCR检测，重组菌中能够扩增出660bp的含有GmSKP1基因序列的片段(图1)，测序结果表明该序列中含有序列一致的GmSKP1基因，即获得了GmSKP1基因序列的植物表达载体。
1.2大豆毛状根的转化
(1)选取健康且均质的大豆种子Williams 82，播种于无菌的人工土壤中，置于24-26℃的生长室生长6-7天，至其苗高4-5cm。
(2)截取大豆双子叶以下4cm处，用蒸馏水洗去上面的泥土。再用小枪头在大豆子叶节处及每隔1.5cm处扎孔，刮取提前划好线的农杆菌反复填充扎孔处，为提高转化效率可在次日再次填充菌落；
(3)保湿系统中培养侵染过的大豆幼苗，保持保湿系统温热湿润的环境下。等到毛状根长到10cm左右即可转移到水培盆或者土培盆中继续生长20天。
实施例2转基因复合体植株的鉴定
2.1基因组水平鉴定
采用植物基因组DNA快捷提取试剂盒，提取两组转基因大豆毛状根的DNA。详细步骤参考试剂盒说明书。用pEGAD载体通用引物进行转基因毛状根的PCR鉴定。PCR反应体系如表1所示，反应程序如表2所示，所用通用引物如表3所示。
表1 PCR反应体系
表2 PCR反应程序
表3通用引物
pEGAD-F：5'-CATGGTCCTGCTGGAGTTCGTG-3'
pEGAD-R：5'-AAATGTTTGAACGATCGGGGAAATTC-3'
2.2转录水平鉴定
从转基因大豆毛状根中提取RNA，具体操作步骤参见试剂盒说明书。将毛状根RNA反转录成cDNA，以cDNA为模板进行qRT-PCR检测，qRT-PCR反应体系如表4所示，反应程序如表5所示，实验所用引物如表6所示。
表4 qPCR反应体系
表5 qPCR反应程序
表6所用引物
nGmSKP1qPCR-F:5'-GGCTGCGAACTACTTGAACATC-3'
nGmSKP1qPCR-R:5'-TTTCCCGACGAACTTCCTCT-3'
在所有GmSKP1-pEGAD256-K599转基因大豆复合体植株中随即选取5棵，提取其毛状根中的基因组DNA。将K599大豆毛状根基因组DNA为阴性对照，以DNA为模板进行PCR检测(图2)，利用载体通用引物进行PCR扩增，结果表明能够从转化的毛状根中扩增出GmSKP1基因，说明已经获得了转GmSKP1基因的大豆毛状根。
为进一步鉴定转基因大豆复合体植株中GmSKP1的转录水平，在所有GmSKP1-pEGAD-K599转基因大豆复合体植株和K599大豆复合体植株中各选取3棵，提取其毛状根中RNA，反转成cDNA，用qPCR方法检测GmSKP1基因的表达情况。
结果显示，转基因毛状根中GmSKP1基因的表达量均显著高于对照植株(图3)，说明已经获得了过表达GmSKP1基因的转基因大豆毛状根。
实施例3转基因复合体大豆的表型鉴定
将GmSKP1-pEGAD-K599转基因复合体大豆和对照植株在水培盆中正常培养25天后，将两组植株毛状根浸没在同样的含有10％PEG的水环境中培养4-6天后，期间每天观察表型变化并统计两组植株的存活率。
结果发现，GmSKP1-pEGAD-K599转基因复合体大豆叶片的萎蔫和失绿程度高于对照植株叶片(图4)，在10％PEG模拟干旱胁迫的处理下，GmSKP1-pEGAD-K599转基因复合体大豆植株在胁迫后的第6天，存活率为23.3％，而对照组植株的存活率为41.66％(图5)。以上结果说明，过表达GmSKP1增加了复合体大豆对干旱胁迫的敏感性。
实施例4转基因复合体大豆干旱胁迫下的生理机制检测
(1)在进行生理指标测定前，需要准备正常生长20天的各株系大豆叶片和干旱胁迫后一周左右大豆叶片，每株各取0.2g叶片。
(2)使用南京建成生物提供的试剂盒来检测叶片中的脯氨酸和可溶性糖含量。
(3)大豆叶片置于研钵中，液氮速冻后快速捣碎，将捣碎后的大豆叶片转入离心管中，后续步骤参照试剂盒说明书，设置三个重复。
测定了10％PEG胁迫前后，GmSKP1转基因复合体大豆和对照大豆叶片中的脯氨酸和可溶性糖含量。检测的结果显示，在10％PEG胁迫之前，GmSKP1-pEGAD-K599转基因复合体大豆叶片中的可溶性糖和脯氨酸均与对照组植株没有差异；在干旱胁迫下，GmSKP1-pEGAD-K599转基因复合体大豆叶片中的可溶性糖和脯氨酸显著低于对照植株(图6)，这说明过表达GmSKP1减少转基因大豆可溶性糖和脯氨酸的累积，减少渗透调节物质的含量，降低转基因大豆的耐旱性。
实施例5 GmSKP1-RNAi转基因复合体大豆干旱胁迫下的表型鉴定及生理机制检测
根据GmSKP1的编码区序列构建RNAi载体，将GmSKP1-RNAi-pCAMBIA3301重组质粒转化发根农杆菌K599，并转化大豆，获得抑制GmSKP1基因的转基因大豆毛状根，进行DNA和RNA水平鉴定转基因阳性毛状根。
将GmSKP1-RNAi-pCAMBIA3301-K599转基因复合体大豆和对照植株在水培盆中正常培养25天后，将两组植株毛状根浸没在同样的含有10％PEG的水环境中培养4-6天后，期间每天观察表型变化并统计两组植株的存活率。
结果发现，GmSKP1-RNAi-pCAMBIA3301-K599转基因复合体大豆叶片的萎蔫和失绿程度低于对照植株叶片，在10％PEG模拟干旱胁迫的处理下，GmSKP1-RNAi-pCAMBIA3301-K599转基因复合体大豆植株在胁迫后的第6天，存活率高于对照组植株。以上结果说明，抑制GmSKP1增加了复合体大豆对干旱胁迫的耐受性。
测定了10％PEG胁迫前后，GmSKP1-RNAi转基因复合体大豆和对照大豆叶片中的脯氨酸和可溶性糖含量。检测的结果显示，在10％PEG胁迫之前，GmSKP1-RNAi-pCAMBIA3301-K599转基因复合体大豆叶片中的可溶性糖和脯氨酸均与对照组植株没有差异；在干旱胁迫下，GmSKP1-RNAi-pCAMBIA3301-K599转基因复合体大豆叶片中的可溶性糖和脯氨酸显著高于对照植株，这说明过表达GmSKP1增加转基因大豆可溶性糖和脯氨酸的累积，增加渗透调节物质的含量，提高转基因大豆的耐旱性。
序列表
<110> 新疆农业大学
<120> GmSKP1基因在负调控大豆干旱胁迫应答中的应用
<160> 1
<170> PatentIn version 3.3
<210> 2
<211> 468
<212> DNA
<213> GmSKP1基因
<400> 1
atgtcgtcgg cgaagaagat cacactgaag agttcggacg gcgaggcttt cgaggtggac 60
gaggcggtgg cgctggagtc tcagacgata aagcacatga tcgaggacga ctgcgccgac 120
agcggcatcc ctctgccgaa cgtgacgagc aagatcctgg cgaaggtgat cgagtactgc 180
aagaagcacg tcgaggccgc gaatcccgaa gacaaaccct ccgaggacga tctcaaagcc 240
tgggacgccg atttcgtcaa ggtcgaccag gccacgctct tcgatcttat cctggctgcg 300
aactacttga acatcaagag cctgctggac cttacatgcc aaactgtagc cgacatgatc 360
aaggggaaga ctcccgagga aattcgcaag acctttaaca ttaagaatga cttcacccct 420
gaggaagaag aggaagttcg tcgggaaaat caatgggcat ttgaatga 468
<210> 2
<211> 155
<212> PRT
<213> GmSKP1基因
<400> 2
MSSAKKITLK SSDGEAFEVD EAVALESQTI KHMIEDDCAD SGIPLPNVTS KILAKVIEYC 60
KKHVEAANPE DKPSEDDLKA WDADFVKVDQ ATLFDLILAA NYLNIKSLLD LTCQTVADMI 120
KGKTPEEIRK TFNIKNDFTP EEEEEVRREN QWAFE 155
Claims (2)
1.GmSKP1基因在负调控大豆干旱胁迫应答中的应用，其特征在于，GmSKP1基因的核苷酸序列如SEQ ID NO：1所示。
2.一种制备耐干旱胁迫大豆的方法，其特征在于，通过对GmSKP1基因的编码区进行基因编辑或RNAi抑制，致使GmSKP1基因功能丧失，从而产生抗旱大豆植物，其中，GmSKP1基因的核苷酸序列如SEQ ID NO：1所示。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102363785A (zh) * | 2011-10-31 | 2012-02-29 | 南京农业大学 | 棉花有丝分裂s期激酶蛋白相关基因skp1及其应用 |
| CN107022011A (zh) * | 2016-02-01 | 2017-08-08 | 中国科学院遗传与发育生物学研究所 | 一种大豆转录因子GmDISS1及其编码基因与应用 |
| CN110577956A (zh) * | 2019-10-31 | 2019-12-17 | 吉林农业大学 | 大豆sHSP26基因及其应用 |
| CN112626069A (zh) * | 2020-12-29 | 2021-04-09 | 新疆农业大学 | 大豆gma-miR4359b基因、其表达载体、制备方法及其应用 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102041248A (zh) * | 2009-10-20 | 2011-05-04 | 中国科学院遗传与发育生物学研究所 | 植物耐逆性相关蛋白GmSIK1及其编码基因与应用 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102363785A (zh) * | 2011-10-31 | 2012-02-29 | 南京农业大学 | 棉花有丝分裂s期激酶蛋白相关基因skp1及其应用 |
| CN107022011A (zh) * | 2016-02-01 | 2017-08-08 | 中国科学院遗传与发育生物学研究所 | 一种大豆转录因子GmDISS1及其编码基因与应用 |
| CN110577956A (zh) * | 2019-10-31 | 2019-12-17 | 吉林农业大学 | 大豆sHSP26基因及其应用 |
| CN112626069A (zh) * | 2020-12-29 | 2021-04-09 | 新疆农业大学 | 大豆gma-miR4359b基因、其表达载体、制备方法及其应用 |
Non-Patent Citations (3)
| Title |
|---|
| "PREDICTED: Glycine max SKP1-like protein 1A (LOC100809769), transcript variant X2, mRNA"，Accession Number:XM_003517112.5;genbank;《GenBank》;第1-2页 * |
| "Soybean F-Box-Like Protein GmFBL144 Interacts With Small Heat Shock Protein and Negatively Regulates Plant Drought Stress Tolerance";Keheng Xu et al.;《Frontiers in Plant Science》;第13卷;第1-13页 * |
| "大豆热激转录因子GmHsFA1对高温和干旱信号的表达反应";吴广锡 等;《分子植物育种》;第11卷(第1期);第62-71页 * |
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