林业科学 ›› 2020, Vol. 56 ›› Issue (10): 70-82.doi: 10.11707/j.1001-7488.20201008
梁璧,张佳琦,任飞,胡恒康,徐川梅,胡渊渊,黄有军,娄和强,张启香*
收稿日期:
2019-11-18
出版日期:
2020-10-25
发布日期:
2020-11-26
通讯作者:
张启香
基金资助:
Bi Liang,Jiaqi Zhang,Fei Ren,Hengkang Hu,Chuanmei Xu,Yuanyuan Hu,Youjun Huang,Heqiang Lou,Qixiang Zhang*
Received:
2019-11-18
Online:
2020-10-25
Published:
2020-11-26
Contact:
Qixiang Zhang
摘要:
目的: 内根-贝壳杉烯氧化酶(KO)是赤霉素(GAs)生物合成途径关键酶,也是植物GAs生物合成抑制剂多效唑的靶酶,若GAs生物合成途径中在KO基因位点发生阻塞,会影响植株的正常生长。本研究克隆山核桃CcKO基因(Caca0733S0131)及其启动子并进行表达分析,以利于进一步探究山核桃CcKO基因在植物生长和发育过程中,尤其是与株高调控相关的生物学功能,从而助力山核桃优新种质的创制。方法: 以山核桃体细胞胚为试验材料,采用同源重组及PCR扩增技术,克隆获得山核桃KO的基因及启动子序列,并进一步构建了具有强启动子的35S∷CcKO∷GFP过表达载体和CcKOpro∷GUS启动子表达载体;利用BLAST在线工具得到该基因编码的氨基酸序列,并对其进行生物信息学分析和同源性分析;进一步通过农杆菌介导法将启动子表达载体及过表达载体转入核桃体细胞胚并进行植株再生试验,分别获得阳性再生植株,从而深入分析山核桃CcKO基因的生物学功能。结果: 通过克隆,获得1条山核桃CcKO开放阅读框,其全长为1 563 bp,共编码520个氨基酸,相对分子量为59.076 kDa。氨基酸同源性分析表明,山核桃CcKO含有细胞色素P450核心功能域FXXGXRXCXG和氨基端的跨膜结构域。BLAST结果表明,山核桃CcKO氨基酸序列与核桃JrKO氨基酸序列同源性为96%,与白梨、苹果、板栗、欧洲栓皮栎的氨基酸同源性分别为71%、79%、77%、76%。对核桃体细胞胚进行遗传转化:启动子表达经GUS染色和PCR验证表明,CcKOpro∷GUS启动子表达载体被成功转入核桃体细胞胚中,且山核桃CcKO基因主要定位于维管束中;过量表达经荧光检测显示及PCR验证表明,35S∷CcKO∷GFP过表达载体被成功转入核桃体细胞胚中;表型分析结果显示,阳性再生植株株高显著高于对照,且山核桃CcKO基因的表达丰度越高,其株高越高;实时荧光定量PCR及相关分析表明,该基因表达具有空间差异,茎中表达量显著高于其他组织部位,且过量表达可使核桃阳性再生植株GAs合成通路下游GA20ox表达量升高而GA2ox表达量下降。结论: 山核桃CcKO基因编码区全长1 563 bp,编码520个氨基酸,该基因所编码的氨基酸序列与核桃JrKO氨基酸序列同源性最高,同源性为96%;启动子表达载体定位结果表明该基因主要定位于维管束。过量表达山核桃CcKO基因的核桃再生植株中,KO mRNA表达量明显升高,植株表现出明显的伸长特征,并且CcKO过量表达可使其GA20ox表达量上调,GA2ox表达量下调。本研究结果可为进一步分析KO基因在山核桃及核桃生长发育过程中的作用提供参考。
中图分类号:
梁璧,张佳琦,任飞,胡恒康,徐川梅,胡渊渊,黄有军,娄和强,张启香. 山核桃贝壳杉烯氧化酶基因CcKO的克隆和表达分析[J]. 林业科学, 2020, 56(10): 70-82.
Bi Liang,Jiaqi Zhang,Fei Ren,Hengkang Hu,Chuanmei Xu,Yuanyuan Hu,Youjun Huang,Heqiang Lou,Qixiang Zhang. Cloning and Expression Analysis of Ent-Kaurene Oxidase Gene CcKo in Carya cathayensis[J]. Scientia Silvae Sinicae, 2020, 56(10): 70-82.
表1
引物序列①"
引物 Primers | 序列 Sequence(5′—3′) | 用途 Usage |
CcKO-1300-F | CTTGTACAGCTCGTC$\underline{{\rm{GGATCC}}}$ATGTCCATGGCCAGCCTAAGCTAC | 扩增 Amplification |
CcKO-1300-R | TCGCCCTTGCTCACCAT$\underline{{\rm{GTCGAC}}}$TTAATTTCTCAGCTTTATCATTGCTTGCATC | 扩增 Amplification |
PCcKO-1301-F | CGGGGATCCTCTAGA$\underline{{\rm{GTCGAC}}}$ATGATGGAGAGACGGAGAG | 扩增 Amplification |
PCcKO-1301-R | TTACCCTCAGATCTA$\underline{{\rm{CCATGG}}}$TTAATAGTACTCTTATAGTTGCGATTTTGG | 扩增 Amplification |
Actin-F | GCCGAACGGGAAATTGTC | 内参 Reference |
Actin-R | AGAGATGGCTGGAAGAGG | 内参 Reference |
QCcKO-F | ACGGATGCAACATGGACAAGAA | 定量 qPCR |
QCcKO-R | CTCTTTCCTCCTCCGAATGCCA | 定量 qPCR |
QJrGA20ox-F | CAAGTGTTTGTAGACGAGTGGC | 定量 qPCR |
QJrGA20ox-R | TGGACTCTTGCTGTTCACCACT | 定量 qPCR |
QJrGA2ox-F | AATCATTACCCTCCATGCCCAG | 定量 qPCR |
QJrGA2ox-R | AAATGATTTGTGGGTCGGTGTG | 定量 qPCR |
GFP-R | TTACTTGTACAGCTCGTCCA | 鉴定 Testing |
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