生物技术通报 ›› 2022, Vol. 38 ›› Issue (10): 164-172.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0055
韩蕾1,2(), 李俊林1,2, 高爱平3, 黄建峰3, 李建召1(), 宋志忠1,3,4()
收稿日期:
2022-01-11
出版日期:
2022-10-26
发布日期:
2022-11-11
作者简介:
韩蕾,女,博士,副教授,研究方向:植物养分高效利用及耐逆机理;E-mail:基金资助:
HAN Lei1,2(), LI Jun-lin1,2, GAO Ai-ping3, HUANG Jian-feng3, LI Jian-zhao1(), SONG Zhi-zhong1,3,4()
Received:
2022-01-11
Published:
2022-10-26
Online:
2022-11-11
摘要:
Shaker类型钾通道在植物生长发育中起重要作用,其在果树中的生物学功能依然未知。克隆并揭示杧果SPIK钾通道基因的功能,为研究热带果树Shaker类型钾通道的生物学功能提供基因资源和理论基础。以杧果品种‘桂热82’为材料,利用RACE-PCR技术克隆Shaker类型钾离子通道基因MiSPIK(GenBank ID:OM179914),利用生物信息学手段分析该基因及编码蛋白的序列特征,通过实时荧光定量PCR(RT-qPCR)分析该基因的组织特异性表达特征,及其在转录水平对缺钾、高钾、低温、NaCl和PEG处理等5种非生物胁迫的响应情况,并创制MiSPIK超表达转基因拟南芥株系。结果显示,MiSPIK蛋白的分子量为90 006.83 kD,等电点(pI)为7.59,含有6个跨膜结构,属于不稳定的亲水蛋白。杧果MiSPIK与梨PbrSPIK和拟南芥AtSPIK的氨基酸序列一致性高达59.46%,三者紧密聚为一类,遗传距离最近。在转录水平上,MiSPIK在杧果花粉中特异性表达,并在一年生嫁接苗根部受缺钾或NaCl处理的抑制均显著降低,受高钾、PEG或低温胁迫诱导而显著增强。MiSPIK在拟南芥中表达后增强了转基因株系的钾素富集能力,并促进转基因植株提早抽薹和开花。杧果MiSPIK是花粉中特异表达的Shaker类型钾通道基因,其表达水平易受多种非生物胁迫的调控,可能在杧果钾素营养高效与利用中发挥作用。
韩蕾, 李俊林, 高爱平, 黄建峰, 李建召, 宋志忠. 杧果钾通道基因MiSPIK的克隆、表达与功能分析[J]. 生物技术通报, 2022, 38(10): 164-172.
HAN Lei, LI Jun-lin, GAO Ai-ping, HUANG Jian-feng, LI Jian-zhao, SONG Zhi-zhong. Cloning,Expression and Functional Analysis of Potassium Channel Gene MiSPIK in Mangifera indica[J]. Biotechnology Bulletin, 2022, 38(10): 164-172.
引物名称 Primer name | 引物序列 Primer sequence(5'-3') |
---|---|
GP1-F | TCTTCACCCCTCTGGA(A/G)TT(C/T)GG |
GP1-R | GAGGGGACCGGCC(A/G)TC(A/G)TA(A/C)TC |
GP2-out | CTAATGGTTAATACCAGCAATC |
GP2-in | CAGAAGGAAGTGTTCCTCTATG |
GP3-out | GCTGGTGCAGCCCTTCTGAATCAG |
GP3-in | GACGTAACATACTTGACCCAC |
MiSPIK-F | CCAGTCTAGGCAACAAACCAAG |
MiSPIK-R | CGCCTCCATTGGCTATCAGATCTG |
pHB-MiSPIK-F | GCCGAGCTCATGAAAATAACGTGGCTGAGAA |
pHB-MiSPIK-R | GCGTCTAGATTACAACCCACCATGAGTTTTAC |
MiActin-F | GAGGAGAAACAGAAGCAAGT |
MiActin-R | AATCATCTTGCTTCTCACCC |
表1 本文所用引物序列
Table 1 Primers used in study
引物名称 Primer name | 引物序列 Primer sequence(5'-3') |
---|---|
GP1-F | TCTTCACCCCTCTGGA(A/G)TT(C/T)GG |
GP1-R | GAGGGGACCGGCC(A/G)TC(A/G)TA(A/C)TC |
GP2-out | CTAATGGTTAATACCAGCAATC |
GP2-in | CAGAAGGAAGTGTTCCTCTATG |
GP3-out | GCTGGTGCAGCCCTTCTGAATCAG |
GP3-in | GACGTAACATACTTGACCCAC |
MiSPIK-F | CCAGTCTAGGCAACAAACCAAG |
MiSPIK-R | CGCCTCCATTGGCTATCAGATCTG |
pHB-MiSPIK-F | GCCGAGCTCATGAAAATAACGTGGCTGAGAA |
pHB-MiSPIK-R | GCGTCTAGATTACAACCCACCATGAGTTTTAC |
MiActin-F | GAGGAGAAACAGAAGCAAGT |
MiActin-R | AATCATCTTGCTTCTCACCC |
图1 MiSPIK RACE克隆过程 A:保守区片段扩增(M:DL2000 marker;1:保守区扩增产物);B:末端RACE扩增(M:DL2000 marker;3:3'-RACE扩增产物;4:5'-RACE扩增产物);C:全长CDS扩增(M:DL2000 marker;1:CDS扩增产物)
Fig. 1 RACE cloning process of MiSPIK A:Amplification of conserved domain(M:DL2000 marker. 1:PCR product of conserved domain). B:RACE PCR amplification of both ends(M:DL2000 marker. 3:RACE PCR product of 3'-end. 4:RACE PCR product of 5'-end). C:Amplification of full length CDS(M:DL2000 marker. 1:PCR product of CDS)
图8 异源超表达转基因创制 A:重组表达载体构建示意图;B:T0代转基因株系PCR鉴定
Fig. 8 Generation of heterologous over-expression transg-enic seedlings A:Schematic diagram of recombinant expression vector construction. B:PCR identification of T0 transgenic lines
指标Indicator | 对照Control | T1转基因株系T1 transgenic lines |
---|---|---|
总鲜重Total fresh weight/g | 11.73±1.34 | 16.86±1.38** |
地上部鲜重Fresh weight of shoots/g | 10.53±1.12 | 15.59±1.09** |
根部鲜重Fresh weight of roots/g | 1.20±0.09 | 1.27±0.13 |
总根长Total root length/cm | 19.26±0.21 | 19.34±0.23 |
地上部钾含量K+ concentration of shoots/(g·kg-1 DW) | 33.47±2.89 | 42.56±3.32* |
根部钾含量K+ concentration of roots/(g·kg-1 DW) | 22.18±1.65 | 29.26±2.14** |
表2 T1代转基因株系生理指标分析
Table 2 Physiological indicator analysis of T1 transgenic lines
指标Indicator | 对照Control | T1转基因株系T1 transgenic lines |
---|---|---|
总鲜重Total fresh weight/g | 11.73±1.34 | 16.86±1.38** |
地上部鲜重Fresh weight of shoots/g | 10.53±1.12 | 15.59±1.09** |
根部鲜重Fresh weight of roots/g | 1.20±0.09 | 1.27±0.13 |
总根长Total root length/cm | 19.26±0.21 | 19.34±0.23 |
地上部钾含量K+ concentration of shoots/(g·kg-1 DW) | 33.47±2.89 | 42.56±3.32* |
根部钾含量K+ concentration of roots/(g·kg-1 DW) | 22.18±1.65 | 29.26±2.14** |
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