马铃薯捕光色素结合蛋白基因<i>StCP24</i>的克隆及不同光照处理下的功能分析

doi:10.7668/hbnxb.20195132

摘要/Abstract

摘要：

为了探究马铃薯捕光色素结合蛋白基因的功能,对捕光色素结合蛋白基因StCP24(LOC102586836)进行生物信息学分析,并用农杆菌介导法将其转入马铃薯品种东农310中,获得转基因马铃薯株系。在不同光照强度条件下培育无性系转基因株系和对照,测定植株的生长指标、叶片生理指标、荧光参数和基因相对表达量等,通过超微结构观察明确该基因对叶绿体形态的影响。生物信息学分析结果表明,StCP24蛋白为亲水性蛋白,StCP24基因的启动子中包含光响应、防御和应激反应等元件;系统发育分析表明,马铃薯StCP24基因编码的氨基酸与栽培种番茄中StCP24基因编码的氨基酸亲缘关系最近。在不同光照强度处理下,转基因株系的茎粗、叶面积、叶片质量、根长、叶绿素a含量、叶绿素b含量、Fv/Fm、ETR、ETRmax、qP显著高于野生型对照。StCP24基因的过量表达可以使类囊体质粒片层堆叠得更加紧密且堆叠程度更高。综上,StCP24基因过量表达可以增加叶片中叶绿素a含量、叶绿素b含量,提高光合作用光反应中电子传递速率,促进转基因马铃薯植株生长。

关键词: 马铃薯, StCP24基因, 光照强度, 过量表达

Abstract:

In order to study the role of phototrapping pigment binding protein gene in potato photosynthesis,the phototrapping pigment binding protein gene StCP24 (LOC102586836)was bioinformatics analyzed and cloned into potato variety Dongnong 310 to obtain transgenic potato.The morphological indexes,leaf physiological indexes,fluorescence parameters,and gene relative expression of transgenic and control lines were measured under different light intensities after clonal propagation.The function of StCP24 gene was analyzed.The effect of this gene on chloroplasts was determined by ultrastructural observation.The results of bioinformatics analysis showed that StCP24 protein was a hydrophilic protein,and the promoter of StCP24 gene contained elements such as photoresponse,defense and stress response.Phylogenetic analysis showed that the amino acids encoded by StCP24 gene in potato were closely related to those encoded by StCP24 gene in cultivated tomato.Under different light intensity treatments,StCP24 gene overexpressed potato significantly increased stem diameter,leaf area,leaf weight,root length,chlorophyll a,b content,Fv/Fm,ETR,ETRmax and qP compared with wild-type potato.The overexpression of StCP24 gene can make the lamellar stacking of thylakoid granules more tightly and to a higher degree.In conclusion,the overexpression of StCP24 gene can increase the content of chlorophyll a and b in leaves,improve the electron transfer rate in photo cooperative photoreaction,and promote the growth of transgenic potato plants.

Key words: Potato, StCP24 gene, Light intensity, Overexpression

刘玉霖, 李世伟, 裴雅婷, 高红秀, 唐鑫华, 石瑛. 马铃薯捕光色素结合蛋白基因StCP24的克隆及不同光照处理下的功能分析[J]. 华北农学报, 2024, 39(6): 55-63. doi: 10.7668/hbnxb.20195132.

LIU Yulin, LI Shiwei, PEI Yating, GAO Hongxiu, TANG Xinhua, SHI Ying. Cloning and Functional Analysis of Potato Light-trapping Pigment Binding Protein Gene StCP24 under Different Light Treatments[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 55-63. doi: 10.7668/hbnxb.20195132.

http://www.hbnxb.net/CN/Y2024/V39/I6/55

图/表 13

表1 引物信息

Tab.1 Primer information

引物名称 Primer name	引物序列(5'—3') Primer sequence (5'—3')	用途 Purpose
StCP24-F	CGGGATCCATGGCTACAACATCTGCTGCGGT	基因CDS扩增
StCP24-R	GCTCTAGACAAACCAAGAGCTCCCAGGGGGG
35S+StCP24-F	AATTCGGTCCCCAGATTAGCCT	转基因马铃薯验证
35S+StCP24-R	CAAACCAAGAGCTCCCAGGGGG
StCP241-F	TTGAAATGGTACAGAGAGG	qRT-PCR
StCP241-R	AGAAGTGTGCCAAAAGAGA
Elfα-F	GATGTTGTGCCAAAGGATGT	内参基因
Elfα-R	AACTTGGTCAATGCGAGA

表2 StCP24蛋白质二级结构预测

Tab.2 Prediction of secondary structure of StCP24 protein

二级结构 Secondary structure	氨基酸数目/个 Amino acid number
α-螺旋Alpha helix	99
链的延伸Extended strand	23
β-转角Beta turn	6
无规卷曲Random coil	128

图1 StCP24蛋白质二级结构(A)、三级结构(B)预测

Fig.1 Prediction of secondary structure(A)and tertiary structure(B)of StCP24 protein

图2 StCP24 启动子元件 ARE.厌氧诱导;circadian.调节昼夜节律控制;TCA-element.水杨酸响应;TGA-element.生长素响应;TC-rich repeats.防御和应激响应;ABRE.脱落酸响应;GATA-motif、Sp1、TCCC-motif、chs-CMA2a、G-box.光响应;LTR.低温响应。

Fig.2 StCP24 promoter element ARE.Anaerobic induction;circadian.Regulate circadian rhythm control;TCA-element.Salicylic acid response;TGA-element.Auxin response;TC-rich repeats.Defense and stress response;ABRE.Abscisic acid response;GATA-motif,Sp1,TCCC-motif,chs-CMA2a,G-box.Photoresponse;LTR.Low temperature response.

图3 马铃薯StCP24氨基酸系统进化树

Fig.3 Phylogenetic tree of the amino acid of potato StCP24

图4 质粒PCR电泳和双酶切检测电泳结果 A:M.Marker,1.空载体,2—3.重组质粒;B:1.质粒,2.双酶切后质粒。

Fig.4 Electrophoresis of plasmid PCR and double enzyme digestion A:M.Marker,1.Empty carrier,2—3.Recombinant plasmid;B:1.Plasmid,2.Double enzyme digested plasmid.

图5 转基因马铃薯PCR检测电泳结果 M.Marker;1.阳性对照;2.阴性对照;3.ddH₂O;4—7.35S+CP24引物扩增PCR产物。

Fig.5 Electrophoretic image of PCR detection of transgenic potatoes M.Marker;1.Positive control;2.Negative control;3.ddH₂O;4—7.PCR product of 35S+CP24 primer amplification.

图6 StCP24过量表达株系与WT的生长对比

Fig.6 The growth comparison between StCP24 overexpression strain and WT

图7 转基因与非转基因马铃薯在不同光照条件下表型分析

Fig.7 Phenotypic analysis of transgenic and non-transgenic potatoes under different light conditions

图8 转基因与非转基因马铃薯在不同光照条件下叶绿素分析

Fig.8 Chlorophyll analysis of transgenic and non-transgenic potatoes under different light conditions

图9 转基因与非转基因马铃薯在不同光照条件下荧光参数分析

Fig.9 Fluorescence parameter analysis of transgenic and non-transgenic potatoes under different light conditions

图10 不同光照处理下StCP24的表达量热图

Fig.10 The expression heat map of StCP24 under different light treatments

图11 StCP24过表达与对照马铃薯叶绿体超微结构 C.叶绿体;G.基粒片层。

Fig.11 Chloroplast ultrastructure of StCP24 overexpression and control potato C.Chloroplast;G.Granular lamella.

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马铃薯捕光色素结合蛋白基因StCP24的克隆及不同光照处理下的功能分析

Cloning and Functional Analysis of Potato Light-trapping Pigment Binding Protein Gene StCP24 under Different Light Treatments

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马铃薯捕光色素结合蛋白基因StCP24的克隆及不同光照处理下的功能分析

Cloning and Functional Analysis of Potato Light-trapping Pigment Binding Protein Gene StCP24 under Different Light Treatments

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