| 杨树油菜素内酯合成基因DET2的克隆与功能分析 |
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| 引用本文： | 李泽华,杜娟,贺学娇,赵树堂,刘颖丽,卢孟柱.杨树油菜素内酯合成基因DET2的克隆与功能分析[J].林业科学研究,2020,33(2):85-92. |
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| 作者姓名： | 李泽华 杜娟 贺学娇 赵树堂 刘颖丽 卢孟柱 |
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| 作者单位： | 林木遗传育种国家重点实验室,中国林业科学研究院林业研究所,北京100091;浙江大学生命科学学院,浙江杭州310029 |
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| 基金项目： | 转基因生物新品种培育重大专项2018ZX08020002-005-002。 |
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| 摘 要： | 目的]DET2基因编码一个5α-还原酶,是油菜素内酯(BRs)合成过程中的关键限速基因。研究DET2基因在杨树生长发育中的作用,对于进一步研究油菜素内酯在木本植物中的调控机制有重要意义。方法]从银腺杨84K(Populus alba×P. glandulosa,‘84K’)克隆得到拟南芥AtDET2同源基因PagDET2,利用生物信息学对其进行序列比对、生化特征分析、构建系统发育进化树。通过RT-PCR分析其在杨树中的表达模式。构建由CaMV 35S强启动子驱动的过表达载体,通过农杆菌介导的叶盘转化法转化84K杨,得到PagDET2-OE转基因植株。分析过表达DET2基因对于转基因植株内源BRs含量、植株生长和抗逆的影响。结果]克隆了包含全长编码区PagDET2基因全长,可编码一个长度为257个氨基酸的蛋白质。其蛋白序列与毛果杨、拟南芥、水稻、棉花、大豆、番茄DET2蛋白同源性较高,说明该基因在进化过程中相对保守。PagDET2在84K杨不同组织中均检测到表达,其在茎中的表达较高。通过ELISA检测植物BRs含量发现,过量表达DET2可以显著提高杨树内源BRs含量。过量表达DET2基因,可以导致转基因植株的高生长,但对盐胁迫更加敏感。结论]DET2基因作为BRs合成的关键基因,在杨树中过量表达可以显著提高内源BRs含量,促进植株增高。DET2转基因植株的获得为进一步分析BR参与木本植物生长发育的调控机制奠定了基础。
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| 关 键 词： | DET2 银腺杨84K 过量表达 油菜素内酯 高生长 |
Cloning and Characterization of Brassinosteriod Biosynthesis-related Gene DET2 in Poplar |
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| LI Ze-hua,DU Juan,HE Xue-jiao,ZHAO Shu-tang,LIU Ying-li,LU Meng-zhu.Cloning and Characterization of Brassinosteriod Biosynthesis-related Gene DET2 in Poplar[J].Forest Research,2020,33(2):85-92. |
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| Authors: | LI Ze-hua DU Juan HE Xue-jiao ZHAO Shu-tang LIU Ying-li LU Meng-zhu |
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| Affiliation: | (State Key Laboratory of Tree Genetic and Breeding,Research Institute of Forestry,Chinese Academy of Forestry,Beijing 100091,China;College of Life Sciences,Zhejiang University,Hangzhou 310029,Zhejiang,China) |
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| Abstract: | Objective] Brassinosteriods(BRs)as essential plant hormones play crucial roles in plant growth and development. DET2, a 5α-reductase, is considered to catalyze a major rate-limiting in BRs biosynthesis. Study on PagDET2 gene is useful to understand the role of BRs in woody plants. Method] In this study, PagDET2, a homologus gene of Arabidopsis AtDET2, was isolated from Poplar 84 K(Populus alba × P. glandulosa, '84 K’). Bioinformatic method was used to sequence alignment and analyze the basic physical, chemical characteristics and evolutionary relationship. Tissue expression patterns in poplar was analyzed by RT-PCR. The over-expression vector was constructed and transformed into poplar 84 K. PagDET2-OE and wild-type plants were used to measure the content of BRs in vivo and analyze the role of DET2 in plant growth and stresses resistance. Result] The CDS of PagDET2 gene was 774 bp, encoding a 257 amino acid protein. The protein sequence of PagDET2 protein were conserved among Populus trichocarpa, Arabidopsis thaliana, rice, cotton, soybean and tomato. Tissue specific expression analysis showed that PagDET2 was detected in root, leaf tissue and stem tissue. Stem tissue of internode 1 to 3 showed the highest expression level among these tissues. The BRs content of poplar was measured by ELISA. DET2 could significantly increase the endogenous BRs content of poplar. Overexpression of DET2 gene can promote plant growth and be more sensitive to salt stress. Conclusion] Overexpression of PagDET2 can significantly increase the endogenous BRs content and increase plant growth. PagDET2 may also be involved in the wood formation and internode elongation. |
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| Keywords: | DET2 Populus alba x P glandulosa ‘84K’ overexpression brassinosteriod height growth |
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