菠萝类纤维素合成酶CslD家族基因鉴定及AcoCslD2a功能分析

doi:10.16420/j.issn.0513-353x.2021-0518

摘要/Abstract

摘要：

从菠萝基因组中鉴定出8个类纤维素合成酶（cellulose synthase-like,Csl）CslD家族基因,其分布于7条染色体及1条Scaffold片段中,外显子数3 ~ 5;依据进化关系将其划分为3个亚组,其中亚组Ⅲ成员数在菠萝中有明显扩增;结合共线性及表达模式分析等预测了家族成员可能的生物学功能。克隆了AcoCslD2a基因cDNA全长序列,亚细胞定位分析表明,其编码蛋白定位于高尔基体。构建ProAtUbq10和ProAtCslD3启动子驱动的AcoCslD2a的植物表达载体,经根癌农杆菌GV3101介导转化至拟南芥atcsld3根毛缺失突变体,不同载体的转基因结果一致,表明AcoCslD2a能够完全恢复突变体根毛数量至野生型水平,可部分恢复突变体的根毛长度。

关键词: 菠萝, 类纤维素合成酶, CslD, 细胞壁, 根毛

Abstract:

In this study,eight CslD genes were characterized in pineapple genome. Chromosomal mapping showed that they were distributed on seven chromosomes and one scaffold. Three to five exons were identified in these genes. Three subgroups were divided according to the phylogenetic analysis,and the number of Group Ⅲ was significantly increased in pineapple. The potential biological functions of the pineapple CslD gene family members were further predicted,basing on the collinearity and gene expression analysis. The full length cDNA sequence of AcoCslD2a was cloned and the subcellular localization analysis indicated that its encoding protein was localized in Golgi apparatus. Two expression vector for AcoCslD2a gene,which were driven by the promoters including ProAtUbq10 and ProAtCslD3,respectively,were constructed and transformed into the Arabidopsis atcsld3 root hair deficient mutant,mediated by Agrobacterium tumefaciens GV3101. The phenotype analysis for the transgenic lines of different expression vectors both indicated that the defect root hair number phenotype of mutant could be recovered to the wild-type phenotype by AcoCslD2a,while the defect root hair length phenotype could be partially recovered.

Key words: pineapple, cellulose synthase-like, CslD, cell wall, root hair

中图分类号:

S668.3

刘朝阳, 廖志婵, 路鑫鑫, 何业华. 菠萝类纤维素合成酶CslD家族基因鉴定及AcoCslD2a功能分析[J]. 园艺学报, 2022, 49(8): 1650-1662.

LIU Chaoyang, LIAO Zhichan, LU Xinxin, HE Yehua. Identification of CslD Gene Family in Pineapple and Functional Analysis of AcoCslD2a[J]. Acta Horticulturae Sinica, 2022, 49(8): 1650-1662.

图/表 10

表1 本研究中用到的引物序列

Table 1 The primer sequences used in this study

引物用途 Primer use	引物名称 Primer name	引物序列（5′-3′） Primer sequence
基因ORF克隆 ORF Cloning	AcoCSLD2a-F	ATGGCATCGAACAGCGCGCTT
基因ORF克隆 ORF Cloning	AcoCSLD2a-R	TTAAGGGAATGTGAAGGAACC
植物表达载体构建 Plant expression vector construction	EcoRⅠ-ProAtUBQ10-F	GAATTCCGACGAGTCAGTAATAAAC
	BamHⅠ-ProAtUBQ10-R	GGATTCTGTTAATCAGAAAAACTCAG
	EcoRⅠ-ProAtCSLD3-F	GAATTCCACTTGTGTCCTGATACTCTC
	BamHⅠ-ProAtCSLD3-R	GGATTCTGTCTAATAATAACACTAT
	PstⅠ-AcoCSLD2a-F	CTGCAGATGGCATCGAACAGCGCGCTT
	SpeⅠ-AcoCSLD2a-R	ACTAGTTTAAGGGAATGTGAAGGAACC
亚细胞定位载体构建 Subcellular localization vector construction	XbaⅠ-GFP-AcoCSLD2a-F	TCTAGAATGGCATCGAACAGCGCGCTT
亚细胞定位载体构建 Subcellular localization vector construction	SacⅠ-GFP-AcoCSLD2a-R	GAGCTCAGGGAATGTGAAGGAACC

图1 菠萝CslD家族成员的基因结构及对应蛋白序列的保守蛋白结构域

Fig. 1 The gene structure of the pineapple CslD genes and conserved domains of the corresponding protein

图2 菠萝及其他10个物种中CslD蛋白的系统进化树 At：拟南芥;Os：水稻;Zm：玉米;Sb：高粱;Bd：二穗短柄草;Ma：香蕉;Gr：棉花;Prupe：桃;Solyc：番茄;Ptr：杨树。

Fig. 2 Phylogenetic tree of CslD proteins in pineapple and other ten species At：Arabidopsis thaliana;Os：Oryza sativa;Zm：Zea mays;Sb：Sorghum bicolor;Bd：Brachypodium distachyon;Ma：Musa acuminate;Gr：Gossypium raimondii;Prupe：Prunus persica;Solyc：Solanum lycopersicum;Ptr：Populus trichocarpa.

图3 菠萝CslD基因的染色体定位及共线性红色线条代表片段复制的CslD基因对。

Fig. 3 The chromosomal localization and synteny analysis of pineapple CslD genes The red lines indicate the segmental duplicated CslD gene pairs.

图4 菠萝、二穗短柄草和水稻（A）,菠萝、香蕉、玉米和拟南芥（B）CslD家族基因的共线性不同颜色的线条代表菠萝与其他各物种中的共线性CslD基因对。Ac：菠萝;Bd：二穗短柄草;Os：水稻;Ma：香蕉; Zm：玉米;At：拟南芥。

Fig. 4 Synteny analysis of CslD genes from pineapple,Brachypodium distachyon and rice（A）and synteny analysis of CslD genes from pineapple,banana,maize and Arabidopsis（B） Lines with different colors indicated the syntenic CslD gene pairs between pineapple and other species. Ac：Ananas comosus;Bd：Brachypodium distachyon;Os：Oryza sativa;Ma：Musa acuminata;Zm：Zea mays;At：Arabidopsis thaliana.

图5 菠萝CslD基因在菠萝不同组织部位不同发育时期的表达量热图 M：雄蕊;T：花柱;P：花瓣;L：叶;S：茎;R：根;S1 ~ S5分别代表菠萝花及果实的不同发育阶段。

Fig. 5 Heat map of pineapple CslD gene expression in various pineapple tissues during different developmental stages M：Stamen;T：Style;P：Petal;L：Leaf;S：Stem;R：Root;S1-S5 indicate different development stages for flower and fruit of pineapple,respectively.

图6 AcoCslD2a基因cDNA全长的克隆（A）、蛋白结构预测（B）及在菠萝各组织部位的表达特征（C）

Fig. 6 The cDNA full length cloning（A）,protein structure prediction（B）and expression features in different pineapple tissues（C）of AcoCslD2a gene

图7 AcoCslD2a蛋白的亚细胞定位

Fig. 7 The subcellular localization of AcoCslD2a

图 8 拟南芥AcoCslD2a转基因株系（培养7 d）的表型

Fig. 8 The phenotype for the transgenic lines（culture for 7 days）of AcoCslD2a gene

图9 拟南芥AcoCslD2a转基因株系（培养7 d）的平均根毛长度和根毛数量

Fig. 9 The average root hair length and root hair number for the transgenic lines（culture for 7 days）of AcoCslD2a gene

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