CN112028979B - 一种提高黄瓜植株抗低温能力的方法 - Google Patents
一种提高黄瓜植株抗低温能力的方法 Download PDFInfo
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Abstract
本发明涉及一种提高黄瓜植株抗低温能力的方法。实验证明,黄瓜G蛋白α亚基及其编码基因能调控黄瓜种子萌发、幼苗生长及植株抗低温性。实施本发明可以培育黄瓜耐低温品种,以及针对其抗低温的特点采取相应的栽培技术措施,最大化减少其受到的低温危害;还可以提高黄瓜种子萌发率,促进黄瓜幼苗生长;对于缓解或消除设施生产中遭遇的冬季低温,提高产量具有重要的理论意义和实用价值。
Description
本申请是对申请日2018.12.13日,申请号201811525835.3,发明名称“G蛋白α亚基在调控黄瓜种子萌 发、幼苗生长及植株抗低温性中的应用”的发明专利申请的分案申请。
技术领域
本发明涉及生物技术领域,具体涉及G蛋白α亚基在调控黄瓜种子萌发、幼苗生长及植株抗低温性中的 应用。
背景技术
植物G蛋白是由α、β以及γ三类蛋白亚基组成的异源三聚体。G蛋白在单子叶植物如玉米、大豆和水稻, 双子叶植物如拟南芥中广泛存在。尽管AGB1(G-proteinβsubunit,G蛋白β亚基)与GPA1(G-proteinαsubunit, G蛋白α亚基)的表达模式类似,但是它们编码的蛋白在细胞内的定位情况却不尽相同,因此AGB1与GPA1 可能在植物中发挥着不同的功能。
黄瓜作为我国重要的设施蔬菜作物,其在设施生产中遭遇的冬季低温是限制其产量的重要因素。研究 表明,黄瓜在受到冷害胁迫时,处于萌发阶段的种子萌发受到抑制,胚根伸长缓慢;黄瓜幼苗叶片出现下 卷皱缩,萎蔫且叶脉黄化开始出现干枯现象;开花早期植株的生长缓慢,会出现严重的花打顶和化瓜等现 象。本发明通过深入研究黄瓜G蛋白α亚基CsGPA1基因的表达特性和相关功能,探究黄瓜抵抗低温的分子 机制,可以培育耐低温品种,并可针对其抗低温的特点采取相应的栽培技术措施,最大化减少其受到的低 温危害。
发明内容
本发明所要解决的技术问题是如何调控黄瓜种子萌发、幼苗生长及植株抗低温性。
本发明研究发现,黄瓜G蛋白α亚基CsGPA1基因的表达与黄瓜种子萌发、幼苗生长及植株抗低温性具 有相关性,从而提出本发明。
具体而言,本发明提供黄瓜G蛋白α亚基在调控黄瓜种子萌发、幼苗生长和/或植株抗低温性中的应用。
上述应用中,
所述G蛋白α亚基的氨基酸序列:
1)如SEQ ID No.2所示;或者,
2)SEQ ID No.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸残基且具有同等活性的氨 基酸序列。
应当理解,本领域技术人员可根据本发明公开的G蛋白α亚基的氨基酸序列(SEQID No.2),在不影响其 活性的前提下,取代、缺失和/或增加一个或几个氨基酸,得到所述G蛋白α亚基的突变序列。因此,本发 明的G蛋白α亚基还包括SEQ ID No.2所示氨基酸序列经取代、替换和/或增加一个或几个氨基酸,具有与G 蛋白α亚基同等活性的衍生多肽。
具体地,所述多个氨基酸残基包括不超过10个氨基酸残基。
上述G蛋白α亚基可人工合成,也可先合成其编码基因,再进行生物表达得到。
本发明还请求保护编码所述G蛋白α亚基的核酸分子在调控黄瓜种子萌发、幼苗生长及植株抗低温性中 的应用。
具体地,编码所述G蛋白α亚基的核酸分子的核苷酸序列:
1)如SEQ ID No.1所示;或
2)SEQ ID No.1所示核苷酸序列经取代、缺失和/或增加一个或几个核苷酸且编码所述G蛋白α亚基的 核酸分子;
3)在严格条件下与1)或2)限定的核苷酸序列杂交,且编码所述G蛋白α亚基的核酸分子。
其中,所述核酸分子可以是DNA,如cDNA、基因组DNA或重组DNA;所述核酸分子也可以是RNA, 如mRNA或hnRNA等。
本领域普通技术人员可以很容易地采用已知的方法,例如定向进化和点突变的方法,对本发明的G蛋 白α亚基的核苷酸序列进行突变。那些经过人工修饰的,具有与本发明分离得到的G蛋白α亚基的核苷酸序 列75%或者更高同一性的核苷酸,只要编码G蛋白α亚基且具有G蛋白α亚基功能,均是衍生于本发明的核 苷酸序列并且等同于本发明的序列。
本发明中,术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的SEQ ID No.2所示 的氨基酸序列组成的蛋白质的核苷酸序列具有75%或更高,或85%或更高,或90%或更高,或95%或更高 同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间 的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
为解决上述问题,本发明还提供了培育转基因黄瓜的方法。
本发明所述培育转基因黄瓜的方法,包括将编码所述G蛋白α亚基的核酸分子导入黄瓜中,得到转基因 黄瓜的步骤;所述转基因黄瓜与所述受体黄瓜相比种子萌发率提高、幼苗生长增加和/或植株抗低温性提高。
本发明中,所述“将编码所述G蛋白α亚基的核酸分子导入黄瓜中”可通过向受体黄瓜中导入重组载体实 现;所述重组载体可为向载体插入编码所述G蛋白α亚基的核酸分子得到的重组质粒。所述重组载体具体可 为重组质粒pBI121-CsGPA1。所述重组质粒表达SEQID No.2所示的G蛋白α亚基。
为解决上述技术问题,本发明还提供了一种黄瓜育种方法。
本发明所述黄瓜育种方法,可包括如下步骤:增加黄瓜中所述G蛋白α亚基的含量或活性,从而提高黄 瓜种子萌发率、促进幼苗生长和/或提高植株抗低温性。
本发明还请求保护黄瓜低温调节蛋白CsWCOR413PM和所述G蛋白α亚基(CsGPA1)在调控黄瓜植株 抗低温性中的应用。
所述黄瓜低温调节蛋白CsWCOR413PM的氨基酸序列如SEQ ID No.4所示。
本发明还请求保护编码所述黄瓜低温调节蛋白CsWCOR413PM的核酸分子和编码所述G蛋白α亚基的 核酸分子在调控黄瓜植株抗低温性中的应用。
所述编码黄瓜低温调节蛋白CsWCOR413PM的核苷酸序列如SEQ ID No.3所示。
所述G蛋白α亚基的核酸分子与上文含义相同。
具体地,所述调控黄瓜种子萌发为促进黄瓜种子萌发(例如提高种子萌发率,尤其是促进黑暗条件下 种子的萌发率);所述调控幼苗生长为促进幼苗生长(例如诱导黑暗条件下子叶、下胚轴和主根的生长, 进而促进幼苗的生长);所述调控植株抗低温性为提高植株抗低温性。
本发明还请求保护所述G蛋白α亚基或编码所述G蛋白α亚基的核酸分子在提高黄瓜内源激素水杨酸 (SA)含量上的应用。具体为提高黄瓜下胚轴的水杨酸含量。
本发明研究发现,黄瓜内源基因CsGPA1可以影响黄瓜种子的发育,促进黑暗条件下种子的萌发率, 并通过诱导黑暗条件下子叶、下胚轴和主根的生长,进而促进幼苗的生长。CsGPA1通过改变内源激素水 杨酸(SA)的含量生成正调控细胞伸长,进而抵御黑暗条件对细胞发育的影响。研究发现,黑暗条件下过 表达CsGPA1转基因株系能够促进幼苗皮层、维管系统和薄壁组织内细胞的分化和伸长。同时,CsGPA1能 够调节皮层和薄壁组织内细胞的分化,并正调节黑暗条件下黄瓜早期幼苗下胚轴的发育
本发明研究发现,黄瓜G蛋白α亚基(CsGPA1)参与了低温胁迫响应,在干扰RNAi株系中,低水 平表达量的CsGPA1降低了植株本身对低温的耐受性。
利用分裂泛素酵母双杂和pull down技术进一步研究发现,在低温(≤6℃)胁迫下,黄瓜低温调节蛋 白CsWCOR413PM和CsGPA1相互作用,协同参与了低温胁迫的响应。
本发明所述低温一般是指≤6℃,尤其是指0-6℃。
本发明中,所述转基因黄瓜不仅包含将所述核酸分子转化受体黄瓜得到的第一代转基因植物,也包括 其子代。对于转基因黄瓜,可以在该物种中繁殖所述核酸分子,也可用常规育种技术将所述核酸分子转移 进入相同物种的其它品种,特别包括商业品种中。
所述转基因黄瓜包括种子、愈伤组织、完整植株和细胞。
实验证明,黄瓜G蛋白α亚基及其编码基因能调控黄瓜种子萌发、幼苗生长及植株抗低温性。实施本发 明可以培育黄瓜耐低温品种,以及针对其抗低温的特点采取相应的栽培技术措施,最大化减少其受到的低 温危害;还可以提高黄瓜种子萌发率,促进黄瓜幼苗生长;对于缓解或消除设施生产中遭遇的冬季低温, 提高产量具有重要的理论意义和实用价值。
附图说明
图1表示黄瓜G蛋白α亚基CsGPA1 cDNA的克隆及重组质粒电泳图谱检测结果;其中,图1A为 CsGPA1 cDNA的RT-PCR克隆;图1B为CsGPA1-Teasy重组菌液PCR检测;M表示DNAmarkerⅢ(2000 bp);S2-S10分别为CsGPA1的PCR特异性扩增片段。
图2表示黄瓜G蛋白α亚基CsGPA1基因的组成结构。
图3表示pBI-121质粒图谱(图3A)和表示重组质粒pBI121-CsGPA1结构示意图(3B)。
图4表示pFGC1008载体图谱。
图5表示CsGPA1过量表达结果和获得的干扰植株;其中,A:CsGPA1过量表达载体的PCR鉴定; B:RNAi干扰载体的PCR鉴定;C:野生型,过表达和干扰植株中CsGPA1相对表达量的分析;D:转基 因植株的生长过程,以黄瓜TUA基因为内参,使用三个生物学重复。
图6表示野生型和黄瓜转基因株系的种子萌发率和种子千粒重大小;其中,图A表示野生型和黄瓜转 基因株系在种子萌发萌发后0天和3天的图片。标尺=1cm。图B表示野生型和黄瓜转基因株系24小时和 48小时的萌发率。图C表示不用转基因株系6天的CsGPA1的相对表达。图D表示野生型和黄瓜转基因 株系种子千粒重(g)。图E表示野生型和黄瓜转基因株系种子长度。图F表示野生型和黄瓜转基因株系 种子宽度。胚根长度为3mm视为种子萌发。三次重复,每次重复至少20粒。WT:野生型;OE:CsGPA1 过表达转基因株系;RNAi:CsGPA1干扰株系。
图7表示野生型和黄瓜转基因株系幼苗的形态特征结果;其中,图A为野生型和转基因幼苗生长3天 的表型图片。图B为野生型和转基因幼苗生长6天的表型图片。标尺=1cm。(图A和图B)生长条件: 在黑暗30℃条件下,铺有2层滤纸直径为9.0cm的培养皿,每个培养皿用移液器加入5ml蒸馏水。野生型 (中间),OE(左面),和RNAi(右面)标尺=1cm。胚根伸长值3mm,视为种子萌发。图C表示下胚轴 长度。图D表示子叶表面积。图E表示下胚轴表面积。图F表示茎粗。图G表示根表面积。图H表示根 体积。图I表示根长度。图J表示下胚轴投影面积。图K表示子叶投影面积。图L表示根投影面积。
图8表示黑暗条件下不同转基因幼苗生长6天下胚轴的横切和纵切结果;其中,(A)OE#97,(B)野生 型,(C)RNAi#10干扰株系。(D)OE#97,(E)野生型和(F)RNAi#10干扰株系下胚轴横切的放大图。标尺= 100μm。EP,外韧;IP,内韧;X,木质部;PP,薄壁细胞;BS,维管束鞘。(G)OE#97,(H)野生型,和 RNAi#10干扰株系下胚轴纵切。虚线表示指定的方向。(J)薄壁细胞最外层平均单个细胞面积。(K)每0.5mm2平均细胞数(n=3个可示区域)
图9表示黑暗条件下不同转基因幼苗生长6天子叶和根尖的横切和纵切结果;其中,(A)OE,(B)野 生型,(C)干扰(RNAi)株系。(D)OE株系子叶纵切,(E)野生型株系子叶纵切,(F)干扰(RNAi)株系子 叶纵切。(G)OE株系根尖纵切,(H)野生型株系根尖纵切,(I)干扰(RNAi)株系根尖纵切。标尺=100μm。
图10表示CsGPA1可能通过改变内源激素水杨酸(SA)的含量影响黄瓜幼苗下胚轴的生长结果;其 中,(A)水杨酸(SA)使用HPLC测定黑暗条件下生长6天幼苗的子叶内源水杨酸含量和(B)黑暗条 件下生长6天幼苗的下胚轴内源水杨酸含量。(C),(D),(E)和(F)分别为黑暗条件下生长6天 幼苗的子叶,下胚轴和根中的CsSR1,CsEDS1,CsCBP60g和CsSR1表达量。使用黄瓜CsActin基因作为内 参,三个生物学重复。
图11表示外施用SA(10μmol/L)对CsGPA1干扰株系下胚轴的影响结果。
图12为CsGPA1在SA调节下胚轴生长发育过程的模式图。
图13表示低温处理对野生型黄瓜幼苗CsGPA1表达和野生型以及转基因黄瓜幼苗表型的影响;其中, (A和B)正常生长条件下,低温处理前三叶一心的黄瓜幼苗。(C)低温(6℃)处理60h后WT植株 的第一片、第二片和第三片叶片的表型。(D)低温(6℃)处理60h后RNAi植株的第一片、第二片和 第三片叶片的表型。WT,RNAi分别代表野生型株系和干扰株系;(E和F)干扰转基因株系(RNAi-9和 RNAi-10)的中CsGPA1相对表达量和Western blot的分析;h为小时。使用黄瓜CsActin基因作为内参, 三个生物学重复。
图14表示低温处理对野生型和转基因黄瓜幼苗相关基因表达的影响结果;其中,(A)叶片中CsGPA1 的表达;(B)叶片中CsWCOR413PM的表达;(C)叶片中CsICE1的表达;(D)叶片中CsCBF1的表 达;(E)叶片中CsCBF3的表达;(F)叶片中CsRD-29的表达;(G)叶片中CsHOS1的表达;不同植 株低温处理前和处理后基因的表达水平。WT,RNAi分别代表野生型株系和干扰株系;h为小时。使用黄 瓜CsActin基因作为内参,三个生物学重复。
图15表示低温处理对野生型和转基因黄瓜幼苗叶片膜脂过氧化、抗氧化酶系统活性以及渗透物质的 影响结果;其中,(A)相对电导率;(B)丙二醛含量;(C)脯氨酸含量;(D)可溶性蛋白含量;(E) 超氧化物歧化酶活性;(F)过氧化物酶活性;(G)过氧化氢酶活性;WT和RNAi分别代表野生型株系 和干扰株系;h为小时,每个实验均采用三个生物学重复,通过Duncan方法进行显著性统计检验。
图16表示pBT3-N-GPA1载体构建;其中,(A)Marker;(B)文库重组率鉴定图;(C)Marker;(D)诱饵 载体双酶切鉴定图。M:DNA marker;lane1:Bglll酶切后的质粒;lane2:DNA质粒。
图17表示pTSU2-APP+pPR3-N在各板(SD-trp-leu/SD-trp-leu-his/SD-trp-leu-his-ade)上生长情况。
图18表示分裂泛素酵母双杂交验证低温(6℃)处理下CsGPA1蛋白与CsWCOR413PM蛋白互作;其中, (A)在SD/–Trp–Leu–His+10mmol L 3-AT培养基上画线培养;(B)图A中菌落的β-半乳糖苷酶活性 检测;(C)利用分裂泛素检测CsGPA1和CsWCOR413PM蛋白互作。
图19表示CsWCOR413PM基因序列比对结果。
图20表示GST-CsGPA1考马斯亮蓝染色实验结果。
图21表示GST-CsGPA1和His-CsWCOR413PM蛋白纯化结果。
图22表示Pull down验证GST-CsGPA1和His-CsWCOR413PM蛋白互作。
具体实施方式
以下实施例进一步说明本发明的内容,但不应理解为对本发明的限制。若未特别指明,实施例中所用 的生化试剂均为市售试剂,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
“新泰密刺”黄瓜(Cucumis sativus L.)栽培品种,为本发明用的野生型材料及过表达和干扰(RNAi) 等转基因植株来源。
菌株:构建载体的菌株农杆菌(Agrobacterium tumefacieus)LBA4404、大肠杆菌(Escherichia coli) DH5α来自本实验室。E.coli DH5a用作质粒的转化宿主,购自华越洋生物公司。酵母(Saccharomyces cevevisiae)菌种MY51,用于泛素酵母双杂实验。
载体:pBI121为基因过表达载体,pFGC1008为干扰RNAi载体,pBT3-N为泛素分裂酵母双杂AD载 体,pGEX4T1和pET21a为原核表达载体。
酶及药品试剂与仪器设备:
限制性内切酶购自NEB和Takara公司;RNA、DNA提取试剂盒,DNA凝胶回收试剂盒,组培使用 的MS粉、琼脂糖、植物凝胶等购自华越洋生物公司。
PrimeScriptTM RT反转录试剂盒(RR047A)购自Takara公司,qRT-PCR试剂盒Green PCR Master Mix等相关试剂均购买自大连宝生物工程公司,植物激素以及组培期间使用的各种抗生素购买自莱伯赫斯 生物公司;植物激素水杨酸(SA)购自Sigma公司;其它的常规试剂和药品等均为国产分析纯。
生物试剂:LA Taq DNA polymerase,dNTP均购自NEB公司;胶回收试剂盒购于AXYGEN公司;PCR 合成试剂购自上海生工生物工程公司;GUS染液购于华越洋生物公司;所用引物均由博迈德公司合成转基 因。
试验主要用到仪器设备为摇床、分光光度计、台式离心机、培养箱、PCR仪、水浴锅、-80℃超低温 冰箱、光照培养箱、荧光定量PCR仪和样品粉碎机等。
HPLC内源激素含量测定
1)仪器、试剂以及材料
Agilent 1260Infinity-6420液相色谱-质谱联用仪。甲醇,甲酸,6-苄基腺嘌呤,吲哚乙酸、脱落酸、赤 霉素、玉米素、水杨酸、茉莉酸,C18 SPE固相萃取柱。5%盐酸甲醇溶液:先量取95mL甲醇加入到广口 瓶中,再量取5ml的盐酸加入其中混匀配成。氯仿-甲醇溶液:先量取50mL氯仿溶液加入到广口瓶中, 再量取50mL甲醇溶液加其中混合配成。
2)样品分析方法
色谱条件:色谱柱工作温度35℃;色谱柱型号Hypersil GOLD C18,尺寸100*2.1mm*1.9um;流速设 置为0.2ml每分钟;流动相为30%乙腈+70%水;进样量2.0μl;
质谱条件:离子源为离子喷雾离子化源(ESI),采用正离子方式进行检测;工作温度300℃;离子 喷射电压为5500V;压力为379kPa;源内气体为N2气体;压力:241kPa;气帘气体(N2)压力为69kPa; 扫描方式为多重反应监测(MRM);碰撞气(N2)压力:Medium。
3)样品预处理按常规方法。
4)相对电导率(REC)测定参考Liu et al.(2007)的方法,
5)膜脂过氧化的测定
丙二醛(MDA)含量用COMIN公司的MDA测试盒按照说明书在UV-1800分光光度计上测定。
6)脯氨酸(Pro)和可溶性蛋白(Solution protein)含量的测定
脯氨酸含量的测定参考Shan et al.(2007)的方法。
7)抗氧化酶活性的测定
SOD、POD和CAT分别用COMIN公司的SOD测试盒、CAT测试盒和POD测试盒按照说明书在 UV-1800分光光度计(SHIMADZU)上测定。
实施例1克隆黄瓜中编码的G蛋白α亚基的基因CsGPA1
1)模板的获得
使用RNA提取试剂盒(GT2654),提取野生型黄瓜叶片RNA,保存于超低温冰箱(-80℃),备用。 采用PrimeScript@RT反转试剂盒(RR047A)反转录出cDNA第一链,保存于-20℃备用。
2)人工合成引物(含有酶切位点Sma I和Xba I)
正向扩增引物:GCTCTAGAATGCTGTCTCATTTGAGTAGAAA;
反向扩增引物:TCCCCCGGGTCACAATAACCCAGCCTCA;
以步骤1)获得的cDNA为模板,以上述引物进行PCR扩增,得到基因的目的片段(见图1A),得到可 能的目的条带回收后连接到T载体上,转化大肠杆菌DH5α感受态后,进行蓝白斑筛选,将菌液PCR检 测正确的阳性E.coli菌液进行测序(图1B)分析,结果表明克隆了1个黄瓜G蛋白α亚基基因全长序列 (CsGPA1)。
序列分析表明该基因CsGPA1的核苷酸序列长度为5879bp,编码392个氨基酸,分子量大小为44.6KDa。 CsGPA1含有13个外显子和12个内含子,定位在第四条染色体上(图2)。
黄瓜G蛋白α亚基基因CsGPA1核苷酸序列见SEQ NO.1,黄瓜G蛋白α亚基氨基酸序列见SEQ NO.2。
实施例2转基因黄瓜的获得
(一)CsGPA1转基因黄瓜
1)重组质粒的构建
分别用特异的限制性内切酶(XbaI,Sma I)对实施例1步骤2)的PCR产物(扩增-纯化)和表达载 体质粒pBI121进行双酶切处理;酶切产物回收后用连接酶连接,得到重组质粒pBI121-CsGPA1。pBI-121 质粒(图3A)及重组质粒pBI121-CsGPA1(图3B)的结构示意图见图3。
2)CsGPA1转基因黄瓜的获得
将重组质粒pBI121-CsGPA1转化农杆菌(LBA4404),然后将构建好载体转入到野生型黄瓜愈伤组 织中(参考“Sui et al.,2012”方法),组织培养后得到转基因植株,将幼苗移至无菌土的花盆里进行培养, 获取叶片。另外将幼苗移栽至栽培温室中,定期维护管理,获取转基因种子。
(二)CsGPA1-RNAi转基因黄瓜
人工合成引物:
CsGPA1-RNAi-upstream-F:AGGCGCGCCAGTAGATCGGGTGTTTAAGGTATAC Asc I
CsGPA1-RNAi-upstream-R:GATTTAAATTCACAATAACCCAGCCTCAAA Swa I
CsGPA1-RNAi-downstream-F:GACTAGTAGTAGATCGGGTGTTTAAGGTATAC Spe I
CsGPA1-RNAi-downstream-R:CGGGATCC TCACAATAACCCAGCCTCAAA BamH I
以实施例1步骤1)获得的cDNA为模板,以上述引物进行PCR扩增,得到两个基因的目的片段。使 用对应的2个限制性内切酶分别对两个目的片段和质粒pFGC1008进行双酶切处理,然后纯化回收酶切后 的所需目的片段和质粒片段,使用连接酶将这两个片段连接起来,构建干扰RNAi基因表达载体。将重组 质粒转化农杆菌(LBA4404),然后将构建好载体转入到野生型黄瓜愈伤组织中(参考“Sui et al.,2012”方 法),组织培养后得到转基因植株,将幼苗移至无菌土的花盆里进行培养,获取叶片。另外将幼苗移栽至 栽培温室中,定期维护管理,获取转基因种子。pFGC1008载体见图4。
(三)转基因黄瓜的鉴定
提取转基因黄瓜植株叶片中DNA。根据不同的载体序列设计不同的特异性的引物,进行PCR反应: (过表达植株)T CsGPA1-OE-F:CTCAGAAGACCAAAGGGCA
T CsGPA1-OE-R:CAGATTTCAGAACAGAAGCGT
(干扰表达植株)T CsGPA1-RNAi-F:GAGGACACGCTCGAGTATAAGA
T CsGPA1-RNAi-R:GCACAACAGAATTGAAAGCAAA
鉴定结果见图5。
在DNA水平(图5C)和RNA水平(图5D)上检测阳性转基因植株。通过分析转基因植株中该基 因的表达水平(图5D),发现过表达转基因植株中CsGPA1基因的表达水平明显上调;而在干扰表达转 基因植株中CsGPA1基因的表达水平明显下调,由此证明外源的序列确实被重组到到黄瓜基因组上。
根据基因鉴定结果,分别选取野生型、3个过表达株系(OE-29,OE-80,OE-97)和3个干扰转基因 RNAi株系(RNAi-9,RNAi-10,RNAi-40)作为代表继续进行后续研究。
实施例3黄瓜G蛋白α亚基基因CsGPA1对黑暗条件下黄瓜种子萌发及黄瓜幼苗生长发育的影响
为了明确CsGPA1是否影响黄瓜幼苗早期的生长,选取黄瓜野生型、CsGPA1(OE)过表达和干扰(RNAi) 株系的种子和幼苗为试样材料(图6A)。在黑暗30℃条件下(黄瓜种子萌发适宜条件)对实验材料种 子处理3天后发现,野生型(WT)、CsGPA1(OE)过表达和干扰(RNAi)株系种子的大小和萌发率均 有差异(图6B)。分别统计该条件下处理24个小时的野生型、CsGPA1(OE)过表达和干扰(RNAi)株 系种子萌发率,WT和OE株系的种子全部萌发,萌发率为100%;而RNAi株系种子处理24个小时的萌 发率仅为49.3%,待处理48个小时后萌发率达到91.67%(图6B)。结果表明CsGPA1可能在胚胎发育和 种子萌发过程中起到正调控作用。
为了进一步明确内源CsGPA1在种子发育和幼苗形态的潜在影响。分别测定WT、OE和RNAi株系种 子的生理特征(千粒重和粒长/宽),种子的萌发率和生理特征表明:与野生型(WT)相比,过表达CsGPA1 能够显著促进种子的发育,种子的千粒重更重,种子更大更饱满。因此进一步提高种子的产量(图6D-F)。
同时,比较黑暗30℃条件下生长6天的WT、OE和RNAi株系的形态特征(长,茎粗,表面积,投 影面积和子叶、下胚轴和根的体积)。结果表明:与WT和RNAi株系相比,OE株系幼苗的子叶、下胚 轴和根的表面积,投影面积和体积更大;下胚轴茎粗更粗(图7A-L)。过表达CsGPA1幼苗的子叶更大, 下胚轴更长并有更多的侧根。因此,过表达CsGPA1可以促进黑暗条件下黄瓜幼苗的发育。以上数据表明, 内源CsGPA1可以影响黄瓜种子的发育,并通过诱导黑暗条件下子叶、下胚轴和主根的生长,进而促进幼 苗的生长。根的长度,表面积,投影面积和体积使用WinRHIZO 2007software(LC4800-II LA2400,Saint Foy, Canada)分析。
为了确定CsGPA1是否通过影响细胞分化和伸长进而影响幼苗的生长。避免光照对试验结果的影响, 分别选取能够明显看出表型差异的黑暗条件下生长6天的WT、OE和RNAi株系幼苗的下胚轴为试验材料 (图8)。在显微镜下观察幼苗下胚轴横切和纵切的石蜡切片,分别比较WT、OE和RNAi株系的表皮, 薄壁组织,维管束和外韧细胞的大小。比较了不同株系幼苗下胚轴横切面显示,与WT相比,OE下胚轴 外韧细胞更大。进一步观察发现;与WT幼苗下胚轴相比,OE下胚轴有更多的表皮和木质部细胞,和更 疏松的细胞层(皮层,薄壁组织,维管束和内韧)(图8A,D和G);相反,观察RNAi株系下胚轴的 横切面发现,其外韧细胞更小且表皮和木质部细胞更少(图8B,E和H),伴随更紧密的细胞层(皮层, 薄壁组织,维管束和内韧)(图8C,F,I,J和K)。
进一步在显微镜下观察黑暗条件下生长6天的WT、OE和RNAi株系幼苗子叶和主根石蜡切片的纵切。 结果表明:与WT和RNAi株系主根相比,OE细胞伸长相对快,细胞更大且细胞数目较少;相反,RNAi 主根和子叶细胞分化和伸长速度则较慢。结果见图9A-I。结果表明:可能由于过表达GPA1促进了细胞的 分化和伸长,其株系具有更大子叶、更长下胚轴和更多数量的侧根。而RNAi株系由于降低了细胞的分化 和伸长,株系表现为子叶更小、下胚轴更短和更少数量的侧根。因此,CsGPA1可能是黑暗条件下幼苗细 胞分化和伸长中的正调控因子,通过过表达CsGPA1促进了细胞分化和伸长,进而促进幼苗的生长。
实施例4
为了进一步明确CsGPA1是如何在黑暗中调节黄瓜下胚轴的生长,其是否通过影响黄瓜幼苗内源激素 水平,继续测定并比较黄瓜野生型和转基因株系的内源水杨酸(SA)含量。与WT相比,黄瓜幼苗子叶和 下胚轴水杨酸含量趋势相同,均为OE株系水杨酸含量提高,RNAi株系水杨酸含量降低。同时,WT和 RNAi株系下胚轴的水杨酸含量相似,然而OE株系略有升高。
为了进一步明确是否SA相关基因的表达引起了SA水平的变化,对黑暗条件下生长6天的WT、OE 和RNAi株系幼苗的子叶、下胚轴和根检测SA相关合成基因(CsSID2,CsSR1(CAMTA3),CsCBP60g, CsEDS1)的表达量进行检测。与WT子叶的表达量水平相比,RNAi株系中下胚轴和根的CsSR1均上调, OE株系CsSR1的表达量水平下调。在RNAi株系中子叶、下胚轴和根中,CsSID2,CsCBP60g和CsEDS1 表达量水平均被抑制,OE株系促进了三个基因的表达量,尤其是在OE株系的子叶中(图10A-F)。
为了确定外源水杨酸是否能恢复因缺失CsGPA1的转基因植株抑制黄瓜幼苗生长的现象,萌发实验前, 先使用外源SA(10μmol/L)浸泡RNAi转基因株系的种子4个小时,野生型和RNAi转基因株系用清水 浸泡4个小时作为对照。正常黑暗、30℃条件下,分别测定萌发后3天和6天种子的下胚轴长度。结果 表明:萌发后3天后,使用外源SA浸种后能恢复因为缺失CsGPA1的转基因植株抑制黄瓜幼苗生长的现 象。结果显示:高水平CsGPA1的表达水平对于幼嫩子叶发育是必须的。CsGPA1可能通过改变SA的生 成正调控细胞伸长,进而抵御黑暗条件对细胞发育的影响。结果见图11。CsGPA1在SA调节下胚轴生长 发育过程的模式图见图12。
实施例5黄瓜G蛋白α亚基基因(CsGPA1)调控低温胁迫
参照表达分析的结果,分别选择了两个与野生型相比CsGPA1基因表达水平差异最大的干扰株系作为 研究材料(CsGPA1-RNAi-9和CsGPA1-RNAi-10),待干扰株系的黄瓜幼苗培养至长出3片成熟叶后(图 13A和B),使用持续(白天和黑夜)的低温条件(6℃)对其进行处理来研究CsGPA1的生物学功能(图13)。 研究的结果显示,当遭受持续低温(6℃),60h时,与野生型植株叶片相比,CsGPA1-RNAi干扰的转 基因黄瓜植株在幼苗时期的叶片(第一片、第二片和第三片)出现更加明显的下垂及萎蔫表型,而CsGPA1 过表达的转基因植株叶片形态基本正常。黄瓜幼苗叶片出现明显萎蔫和下垂(图13C和D)。
研究CsGPA1是否在低温响应中发挥调控作用,测定了野生型和干扰(RNAi)株系叶片中CsGPA1、 CsWCOR413PM、低温通路CBF基因及其下游靶基因的表达量(图14)。研究发现,野生型和干扰(RNAi) 株系叶片中CsGPA1和CsWCOR413PM基因的表达量总体均呈现先上升后下降的趋势,且均在低温处理 24h时达到最高(图14A和B);野生型和干扰(RNAi)株系叶片中CsICE1、CsCBF1、CsCBF3和CsRD-29 基因的表达量变化趋势一致,总体均呈先上升后下降的趋势且干扰(RNAi)株系均低于野生型。其中, CsICE1和CsCBF1基因的表达量在低温处理6h达到最高,CsCBF3基因在12h达到最高,而CsRD-29 基因的表达量则在低温处理3h时既达到最高(图14C-F)。CsHOS1基因表达量在低温处理期间呈上升 趋势,在处理60h达到最大,在整个处理期间干扰(RNAi)株系均高于野生型(图14G)。
低温处理过程中,野生型和干扰(RNAi)株系叶片中的相对电导率(图15A)和丙二醛的含量(图 15B)均呈现上升的趋势。与野生型相比,干扰(RNAi)株系叶片中相对电导率和丙二醛的含量均显著增 加,持续低温胁迫对干扰(RNAi)株系幼苗叶片内细胞膜造成的氧化损伤可能更大;相反,干扰(RNAi) 株系叶片中的渗透压调节物(脯氨酸和可溶性蛋白)则显著降低(图15C和D)。同时,对幼苗叶片中抗 氧化酶(SOD、POD和CAT)的活性进行测定发现,抗氧化酶活性整体均呈现逐渐上升的趋势,且干扰 (RNAi)株系叶片中SOD、POD和CAT活性显著低于野生型(图15E,F和G)。结果表明,与野生型 相比,低温胁迫对干扰(RNAi)株系幼苗叶片造成的伤害更大,降低了其抗低温能力。
实施例6黄瓜G蛋白α亚基基因(CsGPA1)与CsWCOR413PM对调控低温胁迫的互作
为了进一步明确黄瓜CsGPA1基因参与低温信号传导的分子作用机制,本研究构建黄瓜分裂泛素酵母 文库,筛选CsGPA1的相互作用蛋白。黄瓜叶片部总RNA的甲醛变性胶电泳结果显示,RNA完整性好,无 DNA及蛋白质污染,满足构建cNDA文库的要求(图16A)。反转录后PCR扩增、酶切并纯化的dscDNA 在1.0%琼脂糖凝胶中呈smear条带,在250~2000bp之间分布比较集中,适合用来构建cDNA文库(图 16B,文库插入片段鉴定)。将cDNA和pPR3-N载体的Sfi I酶切产物进行PCR鉴定(图16D)。
将构建好的pBT3-N-GPA1与pPR3-N转化NMY51酵母菌株,在非选择培养基SD-trp-leu上生长,在选择 培养基SD-trp-leu-his和SD-trp-leu-his-ade上无显著生长,说明诱饵蛋白不存在自激活,背景值微弱,可进行 后续实验(结果见图17)。
用经过自激活验证,表达pBT3-N-GPA1的NMY51酵母菌制备感受态,将文库质粒转入到NMY51 酵母菌体细胞中,然后将混合均匀的转化液均匀涂布在SD/-leu/-trp/-his三缺培养基上,使用封口膜密封培 养皿,将其倒置于30℃的恒温培养箱中培养3到4天,将培养获得的单克隆酵母菌落克隆转接到一个新 的SD/-leu/-trp/-his/-ade/X-gal四缺培养基上培养以观察生长情况,重复三次转接培养。最终得到了11个能 够正常存活并且显色的单克隆酵母菌株。抽提酵母质粒,将获得的11个pPR3-N质粒:pPR3-N-Y2, pPR3-N-Y4,pPR3-N-Y6,pPR3-N-Y8,pPR3-N-Y10,pPR3-N-Y12,pPR3-N-Y17,pPR3-N-Y19,pPR3-N-Y21, pPR3-N-Y22和pPR3-N-Y2-2分别与质粒pBT3-N-GPA1进行一对一回转验证(涂布筛选平板 SD/-Leu/-Trp/-His/-Ade/X-gal),验证结果均为阳性(即在筛选平板上长出蓝色克隆)。说明pPR3-N-Y2, pPR3-N-Y4,pPR3-N-Y6,pPR3-N-Y8,pPR3-N-Y10,pPR3-N-Y12,pPR3-N-Y17,pPR3-N-Y19,pPR3-N-Y21, pPR3-N-Y22和pPR3-N-Y2-2均与CsGPA1互作(图18A和B)。对上述11个质粒进行测序,获得插入片段 序列。经过balst比对,筛选出与低温有关的蛋白黄瓜质膜低温调节蛋白CsWCOR413PM。使用分裂泛素 酵母双杂交的方法,进一步验证CsGPA1是否与CsWCOR413PM互作。酵母双杂交实验显色结果表明, CsGPA1与CsWCOR413PM能够直接互作(图18C)。
利用Pull down技术进一步验证CsGPA1是否与CsWCOR413PM互作。重组质粒菌液PCR鉴定阳性 菌株送至测序公司进行序列测定,测序结果与客户提供序列信息进行比对,序列比对结果如图所示同源性 为100%(图19)。在考马斯亮蓝的实验结果(图20)中可以看出:GST line是使用单独的GST标签蛋白 进行电泳,GST-CsGPA1 line是使用纯化后的融合蛋白GST-CsGPA1进行上样并电泳。与后续的 Immunblotting结果相对应,对His-CsWCOR413PM融合蛋白进行共孵育,以及后续的pull-down试验(图 21)。Input条带是使用His-CsWCOR413PM纯化蛋白进行上样,作为免疫印迹(Immunoblotting-IB)时 的阳性对照条带。在进行pulldown时,使用GST和GST-CsGPA1蛋白同时与His-CsWCOR413PM蛋白进 行孵育,孵育完成后再使用GST beads与蛋白复合体结合,经过清洗等步骤。将样品进行IB,观察蛋白复 合体中是否含有His-CsWCOR413PM蛋白。在GST line没有观察到His-CsWCOR413PM重组蛋白的条带, 说明GST蛋白与His-CsWCOR413PM重组蛋白不结合。在GST-CsGPAl的条带上,观察到了明显的 His-CsWCOR413PM条带,说明GST-CsGPA1重组蛋白和His-CsWCOR413PM重组蛋白存在相互作用。 实验结果见图20、图22。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以 对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所 做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 中国农业科学院蔬菜花卉研究所
<120> 一种提高黄瓜植株抗低温能力的方法
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1188
<212> DNA
<213> 黄瓜(Cucumis sativus L.)
<400> 1
actagtatgc tgtctcattt gagtagaaat atgggcttac tctgcagcag aaatcgtcat 60
tacaacgaac aagatgctga agagaagacg caggctgcag aaatagagag gcggattgaa 120
caagaaacag aggctgaaaa acatatacaa aaacttcttc tgcttggtgc tggagagtct 180
gggaaatcta caatttttaa gcagataaaa ttgttgttcc aaactgggtt tgatgaggca 240
gagcttaaga gctatattcc agtcattcat gcaaatgtgt atcagactat aaaagtatta 300
catgatggtt cgaaggagct tgctcaaaat gataaagagt tcacgaagta tgttttatcc 360
agtgaaaata aggatattgg tgagaaatta tcggatatcg gaggtagatt ggattacccg 420
cgtttgacta gggagcgtgc acaggatata gagactcttt ggaaagatgc tgcgattcag 480
gaaacgtatt ctcgtggaaa tgaactacag gttccagatt gcacacaata tttcatggaa 540
aatttgcaaa gattatctga tgcaaattat attccaacta aggaggatgt actttatgca 600
agagtccgca caactggtgt tgttgaaatc cagtttagcc ccgttggtga aaataaaaag 660
agtggcgaag tatatagact gtttgatgtt ggtggacaga gaaatgagag gagaaaatgg 720
attcatcttt ttgaaggtgt tacagcagta atcttttgtg ctgctattag tgagtatgat 780
caaacacttt ttgaggatga acagaagaac cgaatgatgg agacgaagga actttttgag 840
tgggttctga aacaagagtg ttttgagaaa acgtcattta tgctttttct caacaaattc 900
gatatcttcg agaaaaaggt tctaaaagtc cctctcagtg tgtgtgaatg gttcaatgat 960
tatcagccgg tttcgactgg aaaacaggag atcgagcatg cctatgagtt cgtgaagaaa 1020
aaattcgagg agttatattt taagagcacg gcaccggatc gagtagatcg ggtgtttaag 1080
gtatacagaa ctactgctct tgatcagaaa cttgtaaaga aaacgttcaa gctcgtagat 1140
gaaactttga ggcggcgaaa tctctttgag gctgggttat tgggtacc 1188
<210> 2
<211> 391
<212> PRT
<213> 黄瓜(Cucumis sativus L.)
<400> 2
Leu Ser His Leu Ser Arg Asn Met Gly Leu Leu Cys Ser Arg Asn Arg
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His Tyr Asn Glu Gln Asp Ala Glu Glu Lys Thr Gln Ala Ala Glu Ile
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Glu Arg Arg Ile Glu Gln Glu Thr Glu Ala Glu Lys His Ile Gln Lys
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Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Phe Lys
50 55 60
Gln Ile Lys Leu Leu Phe Gln Thr Gly Phe Asp Glu Ala Glu Leu Lys
65 70 75 80
Ser Tyr Ile Pro Val Ile His Ala Asn Val Tyr Gln Thr Ile Lys Val
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Leu His Asp Gly Ser Lys Glu Leu Ala Gln Asn Asp Lys Glu Phe Thr
100 105 110
Lys Tyr Val Leu Ser Ser Glu Asn Lys Asp Ile Gly Glu Lys Leu Ser
115 120 125
Asp Ile Gly Gly Arg Leu Asp Tyr Pro Arg Leu Thr Arg Glu Arg Ala
130 135 140
Gln Asp Ile Glu Thr Leu Trp Lys Asp Ala Ala Ile Gln Glu Thr Tyr
145 150 155 160
Ser Arg Gly Asn Glu Leu Gln Val Pro Asp Cys Thr Gln Tyr Phe Met
165 170 175
Glu Asn Leu Gln Arg Leu Ser Asp Ala Asn Tyr Ile Pro Thr Lys Glu
180 185 190
Asp Val Leu Tyr Ala Arg Val Arg Thr Thr Gly Val Val Glu Ile Gln
195 200 205
Phe Ser Pro Val Gly Glu Asn Lys Lys Ser Gly Glu Val Tyr Arg Leu
210 215 220
Phe Asp Val Gly Gly Gln Arg Asn Glu Arg Arg Lys Trp Ile His Leu
225 230 235 240
Phe Glu Gly Val Thr Ala Val Ile Phe Cys Ala Ala Ile Ser Glu Tyr
245 250 255
Asp Gln Thr Leu Phe Glu Asp Glu Gln Lys Asn Arg Met Met Glu Thr
260 265 270
Lys Glu Leu Phe Glu Trp Val Leu Lys Gln Glu Cys Phe Glu Lys Thr
275 280 285
Ser Phe Met Leu Phe Leu Asn Lys Phe Asp Ile Phe Glu Lys Lys Val
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Leu Lys Val Pro Leu Ser Val Cys Glu Trp Phe Asn Asp Tyr Gln Pro
305 310 315 320
Val Ser Thr Gly Lys Gln Glu Ile Glu His Ala Tyr Glu Phe Val Lys
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Lys Lys Phe Glu Glu Leu Tyr Phe Lys Ser Thr Ala Pro Asp Arg Val
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Asp Arg Val Phe Lys Val Tyr Arg Thr Thr Ala Leu Asp Gln Lys Leu
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Val Lys Lys Thr Phe Lys Leu Val Asp Glu Thr Leu Arg Arg Arg Asn
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Leu Phe Glu Ala Gly Leu Leu
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<210> 3
<211> 618
<212> DNA
<213> 黄瓜(Cucumis sativus L.)
<400> 3
actagtatgg tgaaacccaa ccacttgaaa atggtgacgg attctgacgc tgccgatctc 60
atttcctctg atctccggga actcggtaac gctgctagaa agcttgctac acacgctgtt 120
aagctcggtg cttcgggttt tactgcttct tttctccaat ggattgcttc ctttgctgct 180
atttacttgt tgattttgga tcggacgaac tggaagacga atatccttac ttcattgttg 240
attccgtaca ttttctttag tcttcccggt gtgatcttcg gttttttcag gggagagttt 300
ggaaaatggg ttgccgtcat tgctgttgtg ctccgtctct tcttcccgcg acgatttcca 360
gattggcttg aattgcctgg agctttgata cttctcattg tggtggctcc aagtttgttt 420
gccaagacca taagaaacga tcccatcgga gaagcaatct gtttgatcat atcatgctat 480
ttactgcaag aacacatcag agcctctggt ggattcagaa actcattcac aaaagcgaat 540
ggcatctcga acaccatcgg catcatcctt ctcttcgttt tccctgtctg ggcactggta 600
ctccgtgttc ttggtacc 618
<210> 4
<211> 202
<212> PRT
<213> 黄瓜(Cucumis sativus L.)
<400> 4
Met Val Lys Pro Asn His Leu Lys Met Val Thr Asp Ser Asp Ala Ala
1 5 10 15
Asp Leu Ile Ser Ser Asp Leu Arg Glu Leu Gly Asn Ala Ala Arg Lys
20 25 30
Leu Ala Thr His Ala Val Lys Leu Gly Ala Ser Gly Phe Thr Ala Ser
35 40 45
Phe Leu Gln Trp Ile Ala Ser Phe Ala Ala Ile Tyr Leu Leu Ile Leu
50 55 60
Asp Arg Thr Asn Trp Lys Thr Asn Ile Leu Thr Ser Leu Leu Ile Pro
65 70 75 80
Tyr Ile Phe Phe Ser Leu Pro Gly Val Ile Phe Gly Phe Phe Arg Gly
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Leu Leu Ile Val Val Ala Pro Ser Leu Phe Ala Lys Thr Ile Arg Asn
130 135 140
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Gln Glu His Ile Arg Ala Ser Gly Gly Phe Arg Asn Ser Phe Thr Lys
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Pro Val Trp Ala Leu Val Leu Arg Val Leu
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<210> 5
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gctctagaat gctgtctcat ttgagtagaa 30
<210> 6
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tcccccgggt cacaataacc cagcctca 28
<210> 7
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aggcgcgcca gtagatcggg tgtttaaggt atac 34
<210> 8
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gatttaaatt cacaataacc cagcctcaaa 30
<210> 9
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gactagtagt agatcgggtg tttaaggtat ac 32
<210> 10
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cgggatcctc acaataaccc agcctcaaa 29
<210> 11
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ctcagaagac caaagggca 19
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cagatttcag aacagaagcg t 21
<210> 13
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gaggacacgc tcgagtataa ga 22
<210> 14
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gcacaacaga attgaaagca aa 22
Claims (4)
1.一种黄瓜育种方法,包括如下步骤:增加黄瓜中黄瓜G蛋白α亚基的含量或活性,从而提高植株抗低温性;所述G蛋白α亚基的氨基酸序列如SEQ ID No.2所示。
2.根据权利要求1所述黄瓜育种方法,其中,编码所述G蛋白α亚基的核酸分子的核苷酸序列如SEQ ID No.1所示。
3.黄瓜低温调节蛋白CsWCOR413PM和黄瓜G蛋白α亚基在调控黄瓜植株抗低温性中的应用;所述黄瓜低温调节蛋白CsWCOR413PM的氨基酸序列如SEQ ID No.4所示;所述G蛋白α亚基的氨基酸序列如SEQ ID No.2所示。
4.根据权利要求3所述的应用,其中,编码所述G蛋白α亚基的核酸分子的核苷酸序列如SEQ ID No.1所示。
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