CN117511971A - 一个簇毛麦蔗糖非酵解型蛋白激酶SnRK2.9-V基因及其所编码的蛋白和应用 - Google Patents
一个簇毛麦蔗糖非酵解型蛋白激酶SnRK2.9-V基因及其所编码的蛋白和应用 Download PDFInfo
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Abstract
本发明公开了一个簇毛麦蔗糖非酵解型蛋白激酶SnRK2.9‑V基因及其所编码的蛋白和应用。SnRK2.9‑V的cDNA序列为SEQ ID NO.1及其编码的氨基酸序列为SEQ ID NO.2。该基因来自二倍体簇毛麦，该基因在簇毛麦中根、茎、叶和籽粒中都有较高的表达量。通过转基因技术获得3棵超表达SnRK2.9‑V转基因植株，其SnRK2.9‑V的表达量是Fielder表达量的9‑28倍，其过量表达正向调控小麦粒宽和千粒重。因此，SnRK2.9‑V可望用于基因工程育种，将其超表达载体pMWB110‑SnRK2.9‑V导入高产小麦品种中，有望提高小麦的粒宽和千粒重。
Description
技术领域
本发明属于基因工程领域，公开了一个簇毛麦蔗糖非酵解型蛋白激酶SNF1(sucrose non-fermental protein kinase1,SNF1)基因SnRK2.9-V及其超表达载体的应用。
背景技术
栽培小麦的近缘物种在长期进化和自然选择过程中，保留了大量的抗病虫害、抗逆、优质等有益基因，是普通小麦品种改良的重要基因来源，因此，研究和利用亲缘物种抗病基因，是改良小麦抗病性的重要途径。簇毛麦属(Dasypyrum或Haynaldia)植物属于禾本科小麦族小麦亚族，是小麦的三级基因资源库，为异花授粉植物，广泛分布于地中海沿岸和高加索地区。包括二倍体一年生簇毛麦(H.villosa或D.villosum，2n＝14，VV)、二倍体多年生簇毛麦(D.breviaristatum，2n＝14，VbVb)和四倍体多年生簇毛麦(D.hordeaceum，2n＝28，VVVV)。二倍体一年生簇毛麦具有许多重要的农艺性状，在长期演变过程中保留了许多普通小麦不具有的优良性状，是小麦改良的优异基因资源。簇毛麦具有抗条锈病、叶锈病、秆锈病、白粉病、黄花叶病、根腐病和瘿螨等多种小麦病害的特性，同时具有耐寒、耐盐碱、抗旱、分蘖力强、生长茂盛、密穗多花和籽粒粗蛋白含量高等优良特性，已经成为小麦遗传改良的优良野生近缘种质资源。南京农业大学细胞遗传研究所已成功将簇毛麦贮藏蛋白基因Glu-V1、Glu-V3及Gli-V1、抗黄花叶病基因Wss1、籽粒软质基因Dina-D1a/Dinb-D1a、抗白粉病基因Pm21转入到普通小麦(De Pace et al.,2001；Zhang et al.,2012b；Zhang etal.,2014；He et al.,2018；Xing et al.,2018；Kozub et al.,2020；Dai et al.,2020)。然而簇毛麦基因组中调节粒型/粒重的关键基因挖掘的相对较少。目前只报道了来自小麦育种骨干亲本6VS/6DL中克隆的DvGW2，降低DvGW2的表达量，可促进粒宽和粒重的增加(Feng et al.,2021)。因此挖掘簇毛麦粒型/粒重基因，克隆并解析其作用机制，对于育种中更好的利用这一种质资源显得非常迫切。
发明内容
本发明的目的是针对现有技术的上述缺陷，提供一个蔗糖非酵解型蛋白激酶SNF1(sucrose non-fermental protein kinase1,SNF1)基因SnRK2.9-V。
本发明的另一目的是提供该基因的超表达载体。
本发明的又一目的是提供该基因和超表达载体的应用。
本发明的目的可通过如下技术方案实现：
SnRK2.9-V(sucrose non-fermenting-1-relatedprotein kinase 2.9)基因，来自二倍体簇毛麦，其核苷酸序列为SEQ ID NO.1。
该基因编码的蛋白质SnRK2.9-V，其氨基酸序列为SEQ ID NO.2。
所述的含有权利要求1所述的SnRK2.9-V基因的超表达载体，优选以pMWB110为出发载体，将所述的SnRK2.9-V基因全长(SEQ ID NO.1)正向插入pMWB110载体的Sac I单酶切位点所得。
含有所述的SnRK2.9-V基因的基因工程菌；优选以农杆菌为宿主菌。
所述的SnRK2.9-V在培育粒重增加小麦品种中的应用。
所述的含SnRK2.9-V基因超表达载体在培育粒重增加小麦品种中的应用。
所述的基因工程菌在培育粒重增加小麦品种中的应用。
有益效果
本发明利用已报道和本实验室获得的小麦及其近缘物种基因组信息，全基因组水平鉴定和比较不同物种SnRK2基因家族数量、分布、结构和系统进化关系，结合表达特征分析，克隆了簇毛麦中与籽粒发育密切相关的蔗糖非酵解型蛋白激酶基因SnRK2.9-V，初步证明SnRK2.9-V正向调控小麦籽粒发育。进一步利用农杆菌遗传转化技术，将SnRK2.9-V基因的超表达载体pMWB110-SnRK2.9-V转化小麦品种Fielder中，获得转基因阳性植株，分子鉴定和抗性鉴定结果表明，超表达该基因可以提高小麦粒宽和粒重，表明该基因在小麦籽粒发育中发挥正向调控功能，为小麦产量育种提供基础。
本发明从簇毛麦中克隆得到了一个小麦籽粒发育基因SnRK2.9-V及其所编码的蛋白质SnRK2.9-V。SnRK2.9-V可用于基因工程育种，将其插入超表达载体pMWB110，得到该基因的超表达载体。将SnRK2.9-V超表达载体导入小麦品种Fielder中，可以提高Fielder的粒宽和千粒重。
附图说明
图1SnRK2.9-V在二倍体簇毛麦不同组织中表达量的qPCR分析X轴：簇毛麦根、茎、叶、穗、种子不同组织；Y轴：SnRK2.9-V基因在不同组织中的表达倍数。
图2SnRK2.9-V基因超表达载体转化Fielder的T0代阳性转基因植株PCR分子鉴定结果第1泳道为marker:DL2000，第2泳道为超表达载体籽粒(Plasmid)，第3泳道为纯水(ddH2O)，第4泳道为超表达受体Fielder，第5泳道为转基因株系分离出来的阴性对照S-2-T0(OE-SnRK2.9-V-T0-2)，泳道6-8依次为阳性转化植株SOE-1-T0(OE-SnRK2.9-V-T0-1)、SOE-5-T0(OE-SnRK2.9-V-T0-5)、SOE-6-T0(OE-SnRK2.9-V-T0-6)。
图3SnRK2.9-V基因超表达载体转化Fielder的T0代阳性转基因植株qPCR分析结果
X轴：Fielder、阴性对照S-2-T0以及上述3棵转基因阳性植株；Y轴：SnRK2.9-V基因在转基因植株中相对于Fielder的表达倍数。
图4SnRK2.9-V基因超表达载体转化Fielder的T1代阳性转基因植株表型分析A：Fielder、阴性对照S-2-T1(OE-SnRK2.9-V-T1-2)以及上述3棵转基因阳性植株SOE-1-T1(OE-SnRK2.9-V-T1-1)、SOE-5-T1(OE-SnRK2.9-V-T1-5)、SOE-6-T1(OE-SnRK2.9-V-T1-6)的生长发育表型；B：Fielder、阴性对照S-2-T1(OE-SnRK2.9-V-T1-2)以及上述3棵转基因阳性植株SOE-1-T1(OE-SnRK2.9-V-T1-1)、SOE-5-T1(OE-SnRK2.9-V-T1-5)、SOE-6-T1(OE-SnRK2.9-V-T1-6)的籽粒表型。
具体实施方式
实施例1SnRK2.9-V基因克隆及组织特异性的表达特征
设计了同源克隆引物P1(GGGATAAGGAGGCGGGGGAT,SEQ ID NO.3)以及P2(AAACACCACAAATACCGCTT,SEQ ID NO.4)，在簇毛麦叶片的cDNA中克隆得到1086bp序列，该序列如SEQ ID NO.1所示。该序列编码361个氨基酸，序列如SEQ ID NO.2所示，将该基因命名为SnRK2.9-V。
把簇毛麦种子播于培养皿中发芽，露白后移栽到水培盒中。待三叶期，取根、茎、叶样，置于-70℃冰箱内保存备用。然后移栽至土培钵中，取三棱期小穗样，置于-70℃冰箱内保存备用。花后20天取籽粒样，置于-70℃冰箱内保存备用。用TRIZOL(Invitrogen)提取簇毛麦根、茎、叶、穗和籽粒的RNA，利用AMV酶(Takara)合成反转录第一链，得到反转录产物。
应用能特异扩增SnRK2.9-V的特异引物P3(TCTGCTGGATGGAAGCAC CG)和P4(CACGTAAAGGGTTACGCCACAT)，对该基因在簇毛麦不同组织样品中进行qPCR分析。PCR反应在qPCR仪(Roche Light Cycler 480，Roche)上扩增。20μl PCR反应体系中含2μl cDNA,10μl2×SYBR EX Taq TM(Tak aRa)，0.4μl引物P1(10μM)和P2(10μM)。扩增参数为：95℃5min，然后95℃10s、60℃30s，72℃15s，共41个循环。反应结束后，计算相对表达量：根据得到的CT值计算目标基因在处理后的不同时间点相对于未处理的相对表达量，即2-△△CT。其中，△△CT＝(CT.Target-CT.Tublin)Time x-(CT.Target-CT.Tublin)Time 0。Time x表示任意时间点，Time 0表示未处理点。结果表明：SnRK2.9-V在根、茎、叶和籽粒中都有较高的表达量。qPCR的结果表明，SnRK2.9-V可能正向调控小麦籽粒发育(图1)。
实施例2 SnRK2.9-V基因超表达载体的构建
利用上述SnRK2.9-V基因克隆载体pMD18T-SnRK2.9-V，以可特异扩增SnRK2.9-V基因的引物对P5(TCCCCGGGTACCGAGCTCATGGAGAGGGGGCCGA)和P6(TCGGGGAAATTCGAGCTCCTACATGGCGTATACTAT)进行PCR扩增，画线序列为Sac I酶切位点序列，回收扩增片段。用Sac I单酶切将扩增目标片断插入到载体pMWB110的35S启动子后面的多克隆位点Sac I单酶切位点之间。由此获得SnRK2.9-V基因超表达载体pMWB110-SnRK2.9-V。
实施例3 SnRK2.9-V基因超表达载体pMWB110-SnRK2.9-V稳定遗传转化
利用农杆菌介导的遗传转化方法(高彩霞等(2015)，专利号：CN201310726478.8)将pMWB110-SnRK2.9-V转化Fielder的幼胚愈伤组织。以小麦品种Fielder受体，开花当天记录开花日期并进行标记，开花后16DAP，选取生长健壮的小麦，剪取麦穗，将种子剥出。使用70％的酒精进行种子消毒，在超净工作台中将种子的胚取出，并切除胚芽，保留胚其余部分即为未成熟胚，用于下一步实验。使用侵染液(1/10MS+乙磺酸0.1g/L+2,4-二氯苯氧乙酸5mg/L+麦芽糖30g/L)侵染小麦未成熟胚，离心20,000g，30min，弃上清液，得到经过预处理后的胚。将含有外源基因的重组农杆菌悬浮在所述侵染液中得到重悬菌液(OD600＝1.0±0.5)，再将重悬菌液中添加终浓度为200μM的乙酰丁香酮和终浓度为0.1％(质量百分含量，即为质量与体积的百分比)的泊洛沙姆(Pluronic F68，普流尼克F68)，即为侵染用菌液。将预处理后外植体和侵染用菌液混匀，28℃静置1.5h，得到侵染后的外植体。将侵染后外植体在共培养培养基(1/10MS+乙磺酸0.1g/L+2,4-二氯苯氧乙酸5mg/L+麦芽糖30g/L+脯氨酸0.69g/L+抗坏血酸100mg/L+乙酰丁香酮200μM+琼脂糖10g/L，pH5.8)中进行共培养(25℃)，胚和共培养培养基间设有滤纸间隔。4d后，在愈伤诱导培养基(MS+硝酸铵2.4g/L+五水硫酸铜1.25mg/L+乙磺酸1.95g/L+2,4-二氯苯氧乙酸2mg/L+水解酪蛋白1g/L+脯氨酸0.69g/L+麦芽糖40g/L+琼脂8g/L+抗坏血酸100mg/L+特美汀200mg/L，pH5.8)中对共培养胚进行愈伤诱导培养(25℃)，共培养胚以盾片朝上的方式进行诱导培养，得到诱导后愈伤组织。4周后，在分化培养基(MS+硝酸铵2.4g/L+五水硫酸铜1.25mg/L+乙磺酸1.95g/L+谷氨酰胺0.75g/L+蔗糖30g/L+噻苯隆0.5mg/L+草铵膦5mg/L+植物凝胶3g/L+特美汀200mg/L，pH5.8)对诱导后愈伤组织进行分化培养(25℃)，光照强度145-155μmol/m2/s，光周期16/8h。培养4周，得到长出绿苗的愈伤组织，然后在生根培养基(1/2MS+乙磺酸0.5g/L+α-萘乙酸0.5mg/L+蔗糖30g/L+草铵膦5mg/L+植物凝胶3g/L+特美汀200mg/L，pH5.8)上进行生根培养(25℃)，光照强度155μmol/m2/s，光周期16/8h，培养3.5-4.5周，至再生苗长约8cm、根系较健壮时，即可开管炼苗5d，最后洗去根系携带的培养基残渣便可移栽入盆钵，获得再生植株共30棵。
提取所有再生植株基因组DNA，对转化植株利用基因跨内含子内部引物P7(AGATTGCCGATGTGTGGTC)和P8(AGGCTCCTCATACTGGTTGC)进行PCR扩增，鉴定阳性转基因植株。PCR程序：100ng/ul基因组模板，10μM的P7和P8各0.5μl；2.5μl 10×buffer；2.5μl2.5mM的dNTP；1.5μl 25mM的Mg2+；0.25μl(5U/μl)Taq polymerase(TaKaRa)，加水至25μl。PCR反应条件为：94℃预变性3min；94℃30s，55℃45s，72℃30s，30个循环；72℃延伸10min。PCR产物经8％的聚丙烯凝胶电泳检测，其中3株可以扩增308bp的目的条带，鉴定为阳性植株，株系编号依次为：SOE-1-T0(OE-SnRK2.9-V-T0-1)、SOE-5-T0(OE-SnRK2.9-V-T0-5)、SOE-6-T0(OE-SnRK2.9-V-T0-6)(图2)。提取了这3棵阳性植株的RNA，利用qPCR鉴定各个阳性植株中SnRK2.9-V基因的表达情况。结果表明：SOE-1-T0、SOE-5-T0、SOE-6-T0转基因阳性植株的SnRK2.9-V的表达量为Fielder的9-28倍(图3)。
实施例4SnRK2.9-V基因超表达调节籽粒发育的功能研究
连续2年种植于南京农业大学细胞遗传所人工气候室的转基因植株T1代：每个株系在成熟期随机选取30株用于农艺相关性状测定(株高、分蘖数、穗长、小穗数和每穗粒数)。将这30株单株收获后，每株随机取500个小麦籽粒，利用考种仪(托普云农TPKZ-3型)，称取千粒重，同时测定粒长和粒宽相关数据。
以Fielder和转基因阴性株系S-2-T1作为对照，在人工气候室环境下比较转基因T1代的株高、分蘖、穗长、小穗数和籽粒性状等农艺性状。结果表明，2021-2022年和2022-2023年，与Fielder和转基因阴性株系S-2-T1相比，SOE-1-T1、SOE-5-T1、SOE-6-T1在株高、穗长、分蘖、小穗数和每穗粒数也均无显著差异(表1)。同时，分析发现，3个SnRK2.9-V转基因株系在籽粒发育上，与Fielder和转基因阴性株系S-2-T1存在显著差异，其转基因株系的粒宽、千粒重显著增加，但粒长变化不明显(图4A，表1)。2021-2022年，SOE-1-T1、SOE-5-T1、SOE-6-T1与Fielder相比，千粒重分别增加12.80％，16.00％和12.40％，粒宽分别增加9.00％，9.40％和9.70％；与阴性株系S-2-T1相比，千粒重分别增加11.30％，14.50％和11.00％，粒宽分别增加9.00％，9.40％和9.70％(图4B，表2)。在2022-2023年，SOE-1-T1、SOE-5-T1、SOE-6-T1与Fielder相比，千粒重分别增加15.60％，15.30％和13.00％，粒宽分别增加16.20％，16.60％和16.20％；与阴性株系S-2-T1相比，千粒重分别增加15.80％，16.00％和13.30％，粒宽分别增加15.80％，16.10％和15.80％。上述结果说明，SnRK2.9-V过量表达正向调控小麦粒宽和千粒重(表2)。
表1 SnRK2.9-V过量表达对株高、分蘖数、穗长、小穗数和每穗粒数的影响
注：PH：株高；TN：分蘖数；SL：穗长；SNS：小穗数；GNS：每穗粒数。不同字母表示显著性差异分析(P<0.05)。
表2 SnRK2.9-V过量表达对粒长、粒宽和千粒重的影响
注：GL：粒长；GW：粒宽；TGW：千粒重。不同字母表示显著性差异分析(P<0.05)。
Claims (9)
1.一个簇毛麦SnRK2.9-V基因，其特征在于其核苷酸序列为SEQ ID NO.1。
2.权利要求1所述的SnRK2.9-V基因所编码的蛋白质，其特征在于其氨基酸序列为SEQID NO.2。
3.含有权利要求1所述的SnRK2.9-V基因的超表达载体。
4.根据权利要求3所述的SnRK2.9-V基因的超表达载体，其特征在于以pMWB110载体为出发载体，将权利要求1所述的SnRK2.9-V基因正向插入pMWB110载体的Sac I单酶切位点间所得。
5.含有权利要求1所述的SnRK2.9-V基因的基因工程菌。
6.根据权利要求5所述的基因工程菌，其特征在于以农杆菌为宿主菌。
7.权利要求1所述的SnRK2.9-V在培育粒重增加小麦品种中的应用。
8.权利要求3、4所述的含SnRK2.9-V基因超表达载体在培育粒重增加小麦品种中的应用。
9.权利要求5、6所述的基因工程菌在培育粒重增加小麦品种中的应用。
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