马铃薯StSN2的克隆、定位及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-1198

摘要/Abstract

摘要：

Snakin-2（StSN2）属于Snakin/GASA家族,GASAs在调控植物生长发育与抗逆性具有重要作用。克隆StSN2,研究StSN2在马铃薯块茎休眠过程中的功能,为进一步探究StSN2与马铃薯块茎休眠的关系提供理论依据。以马铃薯品种“川芋10号”为材料,克隆StSN2全长CDS序列,并进行生物信息学分析,再利用qRT-PCR分析其组织表达特异性、休眠释放过程表达变化以及对激素的响应情况。结果表明,StSN2开放阅读框为315 bp,编码104个氨基酸,相对分子质量约为11.04 kD,理论等电点为8.91;马铃薯StSN2与番茄、茄子、烟草等茄科植物亲缘关系较近;烟草瞬时表达结果显示该蛋白定位于细胞核、质膜;组织表达分析表明,StSN2在各组织中均有表达,以块茎最高,其次是叶片、根,在茎、花蕾、叶柄最低;进一步研究发现,StSN2表达受ABA、BR以及GA₃调控,尤其是显著响应休眠激素ABA的诱导,并且其表达随休眠的释放而降低,表明StSN2可能参与激素对马铃薯休眠的调控。

关键词: 马铃薯, StSN2, 块茎休眠, 基因克隆, 表达分析, 激素

Abstract:

Snakin-2（StSN2）belongs to the Snakin/GASA family. GASAs play an important role in regulating plant growth and stress resistance. This work aims to clone the StSN2 gene,to study the function of StSN2 in the process of Solanum tuberosum tuber dormancy,and to explore the relationship between StSN2 and S. tuberosum tuber dormancy. The full-length CDS sequence of StSN2 was cloned from potato variety "Chuanyu 10" and analyzed by bioinformatics. The tissue expression specificity,dormancy release process and response to hormone were analyzed by qRT-PCR. The results showed that the open reading frame of the gene was 315 bp,encoding 104 amino acids,the relative molecular mass was about 11.04 kD,and the theoretical isoelectric point was 8.91;the protein was closely related to tomato,eggplant,tobacco and other Solanaceae plants in evolution. The results of tobacco transient expression revealed that the protein was localized in the nucleus and plasma membrane. Tissue expression analysis demonstrated that StSN2 was expressed in all tissues,with tubers being the highest,followed by leaves and roots,and lowest in stems,flower buds,and petioles. Further research discovered that the expression of StSN2 was regulated by ABA,BR and GA₃,especially in response to the induction of the dormant hormone ABA,and its expression decreased with the release of dormancy. In conclusion,StSN2 may be involved in hormone regulation of S. tuberosum dormancy.

Key words: Solanum tubersome, StSN2, tuber dormancy, gene cloning, expression analysis, hormone

彭洁, 邓孟胜, 张杰, 刘石锋, 罗李飞, 王西瑶. 马铃薯StSN2的克隆、定位及表达分析[J]. 生物技术通报, 2021, 37(5): 11-18.

PENG Jie, DENG Meng-sheng, ZHANG Jie, LIU Shi-feng, LUO Li-fei, WANG Xi-yao. Cloning,Location and Expression Analysis of Gene StSN2 in Solanum tuberosum[J]. Biotechnology Bulletin, 2021, 37(5): 11-18.

图/表 8

图1 马铃薯总RNA的提取和StSN2的扩增 A：马铃薯总 RNA电泳图;B：StSN2扩增电泳图;C：菌落PCR检测电泳图。M1：DL5000 DNA Marker;M2：DL2000 DNA Marker

Fig. 1 Extraction of total RNA and amplification of StSN2 in S. tuberosum A: Potato total RNA electrophoresis. B: StSN2 amplification electrophoresis. C: colony PCR detection electrophoresis

图2 StSN2的编码序列及氨基酸序列分析

Fig. 2 Coding sequence and amino acid sequence analysis of StSN2

图3 StSN2蛋白二级结构预测紫色部分为无规则卷曲,蓝色部分为α-螺旋

Fig. 3 Prediction of secondary structure of StSN2 protein The purple part is irregularly curled, and the blue part is α-helix

图4 StSN2蛋白三级结构预测 A：StSN1三级结构 B：StSN2三级结构

Fig. 4 Prediction of tertiary structure of StSN2 protein A: StSN1 tertiary structure. B: StSN2 tertiary structure

图5 StSN2系统进化树

Fig. 5 StSN2 phylogenetic tree

图6 亚细胞定位图 a：融合蛋白荧光信号;b：叶绿体荧光通道;c：明场;d：明场与荧光叠加图;e：空载荧光信号;f：叶绿体荧光通道;g：明场;h：明场与荧光叠加图

Fig. 6 Subcellular localization a: Fluorescence signal of fusion protein. b: Chloroplast fluorescence channel. c: Bright field. d: Superposition of bright field and fluorescence. e: No-load fluorescence signal. f: Chloroplast fluorescence channel. g: Bright field. h: Superposition of bright field and fluorescence

图7 马铃薯不同组织中StSN2的表达分析不同小写字母代表差异显著（P<0.05）

Fig. 7 Analysis of StSN2 expression in different tissues of S. tuberosun Different lowercase letters refer to significant differences (P<0.05)

图8 3种不同的激素处理下马铃薯的发芽率及StSN2的表达分析

Fig. 8 Germination rate and StSN2 expression analysis of S. tuberosun treated with three different hormones

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