柠条CkCAD基因的克隆转化及其抗旱功能分析

doi:10.11686/cyxb2020150

摘要/Abstract

摘要：

干旱胁迫严重影响植物的生长发育甚至生存状态，是限制我国西北荒漠植被恢复的主要非生物胁迫因素之一。实验室前期研究发现随黄土高原由南向北降水减少，柠条苯丙烷生物合成是差异表达最显著的代谢途径。本研究遂以柠条苯丙烷合成途径中木质素合成酶基因CkCAD为研究对象，生信分析表明该基因开放阅读框全长1074 bp，编码357个氨基酸；蛋白序列比对发现柠条CkCAD与非洲相思豆、蒺藜苜蓿、大豆和花生的亲缘关系较近，相似度均在80%以上，其中与非洲相思豆ApCAD最为相似。蛋白质偏酸性且为亲水性蛋白，无跨膜结构域，亚细胞定位于细胞质中。通过农杆菌介导法利用过表达载体pCAMBIA1302将柠条CkCAD转入野生型拟南芥中。在筛选获得T₃代纯合阳性植株后进行抗旱性分析。对T₃代过表达CkCAD拟南芥进行实时荧光定量PCR（qRT-PCR）和Western blot检测发现CkCAD及其酶蛋白在拟南芥中呈稳定表达。相较于野生型拟南芥，T₃代过表达CkCAD拟南芥叶脉长度和叶脉密度更大，脉岛长径、脉岛短径更短，脉岛密度更大，叶脉更为发达且木质素含量更高。同时发现干旱处理下T₃代过表达植株的叶片萎蔫程度、丙二醛含量、相对电导率均低于野生型植株，而相对含水量则高于野生型植株。从而证实干旱胁迫下柠条木质素合成酶基因CkCAD可以促进木质素合成进而提高过表达拟南芥植株的抗旱性。

关键词: 柠条, CkCAD, 木质素, 过表达, 抗旱性

Abstract:

Drought stress seriously affects the growth and development and even survival status of plants， which is one of the main abiotic stress factors restricting the restoration of desert vegetation in northwest China. Previous studies in our laboratory have found that phenylpropanoid biosynthesis of Caragana korshinskii was the most significant metabolic pathway for differential expression as precipitation decreases following the Loess Plateau from south to north. This study focused on the lignin synthase gene CkCAD in the phenylpropanoid biosynthesis pathwayof C. korshinskii. A bioinformatics analysis showed that open reading frame of the CkCAD gene is 1074 bp， encoding 357 amino acids. Protein sequence alignment showed that CkCAD is closely related to Abrus precatorius， Medicago truncatula， Glycine max and Arachis hypogaea， with a similarity of more than 80%. It is most similar to the A. precatorius ApCAD. The protein is acidic and hydrophilic， with no transmembrane domain， and its subcellular location is in the cytoplasm. C. korshinskiiCkCAD was transferred into wild-type Arabidopsis thaliana with overexpression vector pCAMBIA1302 by the Agrobacterium-mediated method to obtain positive plants. The drought resistance was analyzed after screening and obtaining T₃ generation homozygous positive plants. It was found by Real-time fluorescence quantitative PCR and Western blot methods that CkCAD and its enzyme protein were stably expressed in T₃ Arabidopsis plants overexpressing CkCAD. Compared with wild type A. thaliana， the A. thaliana T₃ generation overexpressing CkCAD has longer vein length and greater vein density， shorter vein island length and shorter diameter， greater vein island density， more developed veins and higher lignin content. Simultaneous drought treatment revealed that the degree of leaf wilting， malondialdehyde content， and electrolyte leakage of overexpressed plants were lower than those of the wild type plants， while the relative water content was higher than that of the wild type plants. This study confirmed that the lignin synthase gene CkCAD of C. korshinskii can promote lignin synthesis and improve drought resistance of overexpressed A. thaliana plants under drought stress.

Key words: Caragana korshinskii, CkCAD, lignin, overexpression, drought resistance

畅志鹏, 孙莹莹, 李佳阳, 龚春梅. 柠条CkCAD基因的克隆转化及其抗旱功能分析[J]. 草业学报, 2021, 30(3): 68-80.

Zhi-peng CHANG, Ying-ying SUN, Jia-yang LI, Chun-mei GONG. Cloning and transformation of the CkCAD gene in Caragana korshinskii and analysis of its drought resistance function[J]. Acta Prataculturae Sinica, 2021, 30(3): 68-80.

图/表 12

表1 柠条CkCAD转化和定量所用引物

Table 1 Primers for C. korshinskiiCkCAD conversion and quantification in this study

引物名称Primer name	引物序列Primer sequence （5′-3′）
1302-CkCAD-F	GGACTCTTGACCATGATGGGTAGCCTTGAATCGG
1302-CkCAD-R	CTTCTCCTTTACTAGCTGATCAAGTTTACTGCCTTTGAC
Atactin-F	CACTACCGCAGAACGGGAAA
Atactin-R	GCGATGGCTGGAACAGAACC
CkCAD-F	TCTCTCCCCATACACCTACA
CkCAD-R	CCTTCTCTTTCCAAAACTCC
AtCAD-F	TAGAAGCAGGAGAAAAG
AtCAD-R	CAGGAACCATAGGATAA

图1 柠条CkCAD序列扩增条带M为D2000 DNA maker条带；1为扩增CkCAD的DNA片段。M is the D2000 DNA maker band; 1 is the amplified DNA fragment of CkCAD.

Fig.1 Amplified bands of C. korshinskii CkCAD sequence

图2 柠条CkCAD与其他物种系统发育进化树及氨基酸序列比对结果A: 柠条CkCAD与其他物种中同源CAD系统发育进化树C. korshinskii CkCAD and other species homologous CAD phylogenetic tree；B: 柠条CkCAD与其他物种中同源CAD氨基酸序列比对结果C. korshinskii CkCAD and other species homologous CAD amino acid sequence alignment results。MsCAD: 紫花苜蓿肉桂醇脱氢酶M.sativa cinnamoyl alcohol dehydrogenase; MtCAD:蒺藜苜蓿肉桂醇脱氢酶M.truncatula cinnamoyl alcohol dehydrogenase; CaCAD:鹰嘴豆肉桂醇脱氢酶C.arietinum cinnamoyl alcohol dehydrogenase; ApCAD:非洲相思豆肉桂醇脱氢酶A.precatorius arietinum cinnamoyl alcohol dehydrogenase; AhCAD:花生肉桂醇脱氢酶A.hypogaea cinnamoyl alcohol dehydrogenase; VuCAD:豇豆肉桂醇脱氢酶Vigna unguiculata cinnamoyl alcohol dehydrogenase; GmCAD:大豆肉桂醇脱氢酶G.max cinnamoyl alcohol dehydrogenase; GsCAD:野大豆肉桂醇脱氢酶Glycine soja cinnamoyl alcohol dehydrogenase. 灰色部分为氨基酸序列相同部分，黑色部分为氨基酸序列差异部分。The gray part is the same part of the amino acid sequence, and the black part is the difference part of the amino acid sequence.

Fig.2 Phylogenetic tree and amino acid sequence comparison of C. korshinskii CkCAD and other species

图3 柠条CkCAD蛋白质结构及亚细胞定位预测A: 保守结构域分析 Conserved domain analysis; B: 蛋白质二级结构 Protein secondary structure; C:蛋白质三级结构 Protein tertiary structure；D:亚细胞定位预测结果 Subcellular location prediction results.

Fig.3 CkCAD protein structure and subcellular localization prediction of C. korshinskii

图4 pCAMBIA1302-35S::CkCAD过表达载体的构建A: pCAMBIA1302-35S::CkCAD过表达载体示意图；B: pCAMBIA1302-35S::CkCAD过表达载体的酶切鉴定结果。A: pCAMBIA1302-35S:: CkCAD overexpression vector schematic diagram; B: Restriction digestion identification result of pCAMBIA1302-35S::CkCAD overexpression vector.M为D15000 DNA maker; 1为pCAMBIA1302-35S::CkCAD过表达载体Bam HI酶切条带。M is D15000 DNA maker; 1 is the pCAMBIA1302-35S:: CkCAD overexpression vector Bam HI digested band.

Fig.4 Construction of pCAMBIA1302-35S::CkCAD overexpression vector

图5 转CkCAD拟南芥阳性植株的筛选

Fig.5 Screening of CkCAD transgenic Arabidopsis positive plants

图6 柠条CkCAD能够在转基因拟南芥中稳定表达A: CAD基因在野生型和过表达拟南芥株系#1、#3和#4中的定量结果。B: 野生型和过表达拟南芥植株中GFP标签的蛋白免疫印迹结果。CkCAD-GFP为过表达拟南芥株系#1、#3和#4中CkCAD与GFP(绿色荧光蛋白)形成的融合蛋白，GFP为绿色荧光蛋白。WT表示野生型拟南芥，#1、#3、#4表示3个转CkCAD拟南芥株系，**代表差异显著(P<0.01)，下同。A: Quantitative results of CAD genes in wild-type and overexpressing Arabidopsis lines # 1, # 3, and # 4. B: Western blot results of GFP tags in wild-type and overexpressing Arabidopsis plants. CkCAD-GFP is a fusion protein formed by CkCAD and GFP (green fluorescent protein) in overexpressing Arabidopsis strains #1, #3 and #4, GFP is green fluorescent protein. WT represent wild-type Arabidopsis, #1, #3, #4 represent three CkCADArabidopsis lines, the asterisk represents significant difference (P<0.01), the same below.

Fig.6 C. korshinskiiCkCAD can be stably expressed in transgenic Arabidopsis

图7 干旱胁迫下转CkCAD拟南芥植株的生长表型

Fig.7 Growth phenotypes of CkCAD transformed Arabidopsis plants under drought stress

图8 干旱胁迫下野生型及转CkCAD拟南芥植株株高和叶面积的变化趋势

Fig.8 Variation trend of plant height and leaf area of wild-type and CkCAD transformed Arabidopsis plants under drought stress

图9 野生型和转CkCAD拟南芥植株叶脉表型比较

Fig.9 Comparison of leaf vein phenotype between wild-type and CkCAD transgenic Arabidopsis plants

图10 野生型和转CkCAD拟南芥植株叶脉特征分析

Fig.10 Analysis of vein characteristics of wild-type and CkCAD transgenic Arabidopsis plants

图11 干旱胁迫下野生型和转CkCAD拟南芥抗旱性比较分析

Fig.11 Comparison of drought resistance between wild-type and CkCAD-transformed Arabidopsis under drought stress

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