南京林业大学学报(自然科学版) ›› 2021, Vol. 45 ›› Issue (3): 71-78.doi: 10.12302/j.issn.1000-2006.202011014
王培龙(), 杨妮, 张傲然, 唐努尔·塞力克, 李爽, 高彩球*()
收稿日期:
2020-11-07
修回日期:
2021-01-07
出版日期:
2021-05-30
发布日期:
2021-05-31
通讯作者:
高彩球
基金资助:
WANG Peilong(), YANG Ni, ZHANG Aoran, Tangnver•Sailike , LI Shuang, GAO Caiqiu*()
Received:
2020-11-07
Revised:
2021-01-07
Online:
2021-05-30
Published:
2021-05-31
Contact:
GAO Caiqiu
摘要:
【目的】探究刚毛柽柳(Tamarix hispida)植物络合素合酶(phytochelatin synthase,PCS)基因ThPCS1的镉胁迫应答功能。【方法】通过逆转录聚合酶链式反应(RT-PCR)对刚毛柽柳ThPCS1基因进行克隆;通过BioEdit、MEGA 5.0等软件对ThPCS1蛋白进行生物信息学分析;通过实时荧光定量PCR(RT-qPCR)技术分析镉(Cd)胁迫条件下ThPCS1基因在柽柳根和叶中的表达水平;构建pROKII-ThPCS1过表达载体,通过瞬时侵染技术获得转ThPCS1基因柽柳,分析比较转基因和对照柽柳Cd胁迫应答相关生理指标和生理染色情况。【结果】从刚毛柽柳转录组数据中分离出ThPCS1基因的全长转录组序列,预测该基因的开放阅读框为1 581 bp,编码526个氨基酸,编码蛋白质的分子质量为130.75 ku,理论等电点pI为5.0。保守结构域的多重比对分析结果均显示ThPCS1蛋白在N端具有PCS结构域。RT-qPCR结果显示在镉胁迫条件下,ThPCS1基因在柽柳根中被诱导表达,且呈现出组织特异性的表达模式。对镉胁迫处理后的刚毛柽柳ThPCS1转基因植株和对照植株相关生理指标进行测定,结果显示,同对照植株相比,转基因植株的活性氧和镉离子含量降低,细胞膜损伤减小。【结论】刚毛柽柳ThPCS1基因在根中的表达明显受到镉胁迫诱导,可能参与了刚毛柽柳对镉胁迫的应答。本研究初步证明刚毛柽柳ThPCS1基因可能提高了转基因植物的耐镉能力,是一个耐镉分子育种的候选基因。
中图分类号:
王培龙,杨妮,张傲然,等. 刚毛柽柳ThPCS1基因克隆与镉胁迫应答分析[J]. 南京林业大学学报(自然科学版), 2021, 45(3): 71-78.
WANG Peilong, YANG Ni, ZHANG Aoran, Tangnver•Sailike , LI Shuang, GAO Caiqiu. Cloning ThPCS1 gene of Tamarix hispida to improve cadmium tolerance[J].Journal of Nanjing Forestry University (Natural Science Edition), 2021, 45(3): 71-78.DOI: 10.12302/j.issn.1000-2006.202011014.
表1
RT-qPCR和载体构建引物序列"
用途 application | 引物名称 prime name | 引物序列 prime sequences (5'-3') |
---|---|---|
RT- qPCR | ThPCS1-DL-F | CAGCTCTTCACTCTGAGCTGC |
ThPCS1-DL-R | CTCCTCTTGACTTAGACTTG | |
Actin-F | AAACAATGGCTGATGCTG | |
Actin-R | ACAATACCGTGCTCAATAGG | |
α-tubulin-F | CACCCACCGTTGTTCCAG | |
α-tubulin-R | ACCGTCGTCATCTTCACC | |
β-tubulin-F | GGAAGCCATAGAAAGACC | |
β-tubulin-R | CAACAAATGTGGGATGCT | |
基因 克隆 gene clone | ThPCS1-F | CGCGGATCCATGGCGATGGCTGGACTGTAC |
ThPCS1-R | CGGGGTACCAGAGGATAGCGTAGCTGCTGAGTTCTC | |
载体 引物 carrier primers | ThPCS1-T-F | ATGGCGATGGCTGGACTGTAC |
ThPCS1-T-R | AGAGGATAGCGTAGCTGCTGAGTTCTC | |
pROKⅡ-F | AGACGTTCCAACCACGTCTT | |
pROKⅡ-R | CCAGTGAATTCCCGATCTAG |
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