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作物学报

作物学报 ›› 2024, Vol. 50 ›› Issue (4): 932-943.doi: 10.3724/SP.J.1006.2024.34122

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

花生赤霉素3-β-双加氧酶(AhGA3ox)基因家族的全基因组鉴定及表达分析

李海芬(), 鲁清, 刘浩, 温世杰, 王润风, 黄璐, 陈小平, 洪彦彬, 梁炫强()   

  1. 广东省农业科学院作物研究所 / 广东省农作物遗传改良重点实验室 / 国家油料作物改良中心南方花生分中心, 广东广州 510640
  • 收稿日期:2023-07-14 接受日期:2023-10-23 出版日期:2024-04-12 网络出版日期:2023-11-14
  • 通讯作者: * 梁炫强, E-mail: Liangxuanqiang@gdaas.cn
  • 作者简介:E-mail: lihaifen@gdaas.cn
  • 基金资助:
    广东省“十四五”农业科技创新十大重点项目公开竞争计划(2022SDZG05);广东省重点研发计划-现代种业(2020B020219003);广东省重点研发计划-现代种业(2022B0202060004);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-13);国家自然科学基金项目(32001442);国家自然科学基金项目(32172051);广东省基础与应用基础研究基金项目(2021A1515010811);广东省基础与应用基础研究基金项目(2023A1515010098);广东省农业和农村厅科技专项资金项目(2019KJ136-02);广东省海外名师计划项目(207124505346);广东省农业科学院农业优势产业学科团队建设项目(202104TD)

Genome-wide identification and expression analysis of AhGA3ox gene family in peanut (Arachis hypogaea L.)

LI Hai-Fen(), LU Qing, LIU Hao, WEN Shi-Jie, WANG Run-Feng, HUANG Lu, CHEN Xiao-Ping, HONG Yan-Bin, LIANG Xuan-Qiang()   

  1. Guangdong Provincial Key Laboratory of Crop Genetic Improvement / South China Peanut Sub-Center of National Center of Oilseed Crops Improvement / Crops Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, Guangdong, China
  • Received:2023-07-14 Accepted:2023-10-23 Published:2024-04-12 Published online:2023-11-14
  • Contact: * E-mail: Liangxuanqiang@gdaas.cn
  • Supported by:
    Open Competition Program of Top Ten Critical Priorities of Agricultural Science and Technology Innovation for the 14th Five-Year Plan in Guangdong Province(2022SDZG05);Guangdong Provincial Key Research and Development Program- Modern Seed Industry(2020B020219003);Guangdong Provincial Key Research and Development Program- Modern Seed Industry(2022B0202060004);China Agriculture Research System of MOF and MARA(CARS-13);National Natural Science Foundation of China(32001442);National Natural Science Foundation of China(32172051);Guangdong Basic and Applied Basic Research Foundation(2021A1515010811);Guangdong Basic and Applied Basic Research Foundation(2023A1515010098);Technology Special Fund of Guangdong Province Agriculture and Rural Affairs Department(2019KJ136-02);Guangdong Province Overseas Master Teacher Program(207124505346);Agricultural Competitive Industry Discipline Team Building Project of Guangdong Academy of Agricultural Sciences(202104TD)

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摘要：

赤霉素3-β-双加氧酶(gibberellin 3-beta-dioxygenase, GA3ox)是参与赤霉素生物合成的关键酶之一, 可通过影响赤霉素的形成调控植物的生长发育, 目前在花生中尚无系统研究。本研究利用生物信息学方法在花生栽培种基因组数据库中筛选花生GA3ox家族基因, 对鉴定出的7个AhGA3ox在栽培种花生基因组中的分布、结构及进化特征、理化性质、启动子顺式作用元件进行分析, 并利用qRT-PCR技术对其进行花生组织结构表达模式分析, 同时对AhGA3ox家族基因在不同荚果大小的2个花生品系中的表达量进行分析。结果表明, 7个AhGA3ox基因分布在7条染色体上, 均由1个内含子和2个外显子组成。花生AhGA3ox蛋白中均包含1个DIOX_N结构域和1个2OG-FeII_Oxy结构域, 系统进化分析表明与大豆的亲缘关系较近, 在根、茎、叶、花、果针5个组织中呈现出不同表达模式, 不仅在花生果壳不同发育时期的表达量不同, 在2个荚果大小不同的花生品系果壳相同发育时期的表达量也不相同, 但多数发育时期在大果品系中的表达量均显著高于小果品系, 推测该家族基因的表达可能对荚果的形成具有促进作用。

关键词: 花生, GA3ox, 表达分析, 生物信息学, 功能分析

Abstract:

Gibberellin 3-beta-dioxygenase (GA3ox) is one of the key enzymes involved in gibberellin biosynthesis, which can regulate plant growth and development by affecting gibberellin formation. There were no systematic studies in peanut genome. In this study, AhGA3ox family genes were identified from the genome database of cultivated peanut species by bioinformatics, and the distribution, structure, evolutionary characteristics, physical and chemical properties, promoter cis-acting elements were also analyzed. The relative expression pattern of AhGA3oxs in different peanut tissues was analyzed by qRT-PCR, and the relative expression level of in shell of two peanut lines with different pod size were also analyzed. The results showed that 7 AhGA3oxs were distributed on 7 chromosomes of peanut, all of which were composed of 1 intron and 2 exons. All the AhGA3ox proteins contained 1 DIOX_N domain and 1 2OG-FeII_Oxy domain. Phylogenetic analysis showed that they were closely related to GA3ox proteins of soybean,. The AhGA3oxs showed obvious tissue expression specificity in root, stem, leaf, flower and peg. The expression levels of AhGA3oxs were not only different in different development stages of the peanut shell, but also different at the same development stage of the two lines. Interestingly, the relative expression levels in large pod lines were significantly higher than those in small pod lines at most development stages. Therefore, we predict that the gene expression of AhGA3ox gene family may promote the formation of large pod.

Key words: peanut, GA3ox, the relative expression analysis, bioinformatics, functional analysis

表1

qRT-PCR所用引物"

引物名称
Primer name		 引物序列
Primers sequence (5°-3°)		 引物名称
Primer name		 引物序列
Primers sequence (5°-3°)
actin-F CCATTGAGAAGACCTACGA Ahy_A09g046613-F CACATACGTCCACATCCAA
actin-R AATCATGGAAGGCTGGAA Ahy_A09g046613-R TGGTTCCAAGGTACTCATTC
Ahy_A06g030353-F TGGATCAAGTGGTTTCTC Ahy_B06g085507-F GTAGTTTCTCCTTTGGTTCCGAAT
Ahy_A06g030353-R CAAGGCTTTGTCAAGATAC Ahy_B06g085507-R GGCTTTGTCAAGATACTTGTCCTT
Ahy_A07g037374-F TGTCCACATGCGAAGCTA Ahy_B09g094759 -F TCCACATCCAAAGCTAATAGGC
Ahy_A07g037374-R TGGTGCCAAGGTACTCATT Ahy_B09g094759 -R GGTTCCAAGGTACTCATTCCAA
Ahy_A08g040809-F ATTCCCACGCATGGTTTCA Ahy_B08g089553-F CGTTACTCTTTGGCTTATTTCC
Ahy_A08g040809-R CGATGATGGGTATGGATGGAAT Ahy_B08g089553-R CTTCACACTAACCCTACGAAAT

表2

AhGA3ox家族基因蛋白理化性质预测"

基因编号
Gene ID		 CDS大小
CDS size (bp)		 外显子
数目
No. of exons		 亚细胞定位
Subcellular
localization		 等电点
pI		 不稳定
参数
Instability index		 脂溶指数
Aliphatic index		 亲水性
平均数
GRAVY		 带正电荷残基数
Asp + Glu		 带负电荷残基数Arg+Lys
Ahy_A06g030353 1047 2 细胞核 Nucleus 5.53 39.42 82.66 -0.358 46 36
Ahy_A07g037374 1125 2 细胞核 Nucleus 8.11 37.63 86.29 -0.221 31 33
Ahy_A08g040809 1056 2 细胞质 Cytoplasm 7.30 36.94 77.87 -0.434 40 40
Ahy_A09g046613 1068 2 细胞质和细胞核
Cytoplasm and nucleus		 7.68 39.04 87.08 -0.231 30 31
Ahy_B06g085507 1047 2 细胞骨架 Cytoskeleton 5.53 39.09 82.66 -0.370 46 36
Ahy_B08g089553 1062 2 细胞质 Cytoplasm 6.49 37.31 75.79 -0.462 43 39
Ahy_B09g094759 1092 2 细胞核 Nucleus 8.08 39.45 85.71 -0.287 31 33

图1

AhGA3ox基因家族的染色体分布 黑色代表染色体, AhGA3ox家族基因标记在染色体右边, 左侧比例尺表示染色体长度。"

图2

花生AhGA3ox家族基因及编码蛋白的系统发育与结构分析 (A) 利用AhGA3ox的氨基酸序列构建系统发育树。(B) AhGA3ox基因的结构。绿色矩形、黄色矩形和黑色线条分别代表UTR、CDS和内含子。底部比例尺代表UTR、CDS和内含子长度。(C) AhGA3ox蛋白的结构。蓝色和紫色矩形表示DIOX_N结构域, 2OG-FeII_Oxy结构域结构域。底部比例尺表示结构域长度。"

图3

GA3ox基因家族的系统发育分析 浅蓝色背景区域为四倍体野生种花生(Arachis monticola), 绿色区域为栽培种大豆(Glycine max), 蓝色区域为水稻(Oryza sativa), 浅灰色区域为拟南芥(Arabidopsis thaliana), 黄色区域为A基因组二倍体野生种花生(Arachis duranensis), 褐色区域为B基因组二倍体野生花生(Arachis ipaensis), 紫色区域为野生种大豆(Glycine soja), 红色区域为四倍体栽培种花生(Arachis hypogaea L.)。花生、拟南芥、水稻、大豆、野生豆系统发育分析将71个GA3ox蛋白分为4类。利用MEGA-X软件以GA3ox蛋白的氨基酸序列为基础, 采用最大似然法构建系统发育树。Bootstrap=1000。"

图4

GA3ox家族蛋白家族蛋白保守基序分布 Motif分析通过MEME在线分析完成, 不同氨基酸的高度代表可重复性, 底部的比例尺表示基序长度。左侧为蛋白系列名称, 右侧不同颜色分别对应不同的保守基序在系列上的位置。"

图5

AhGA3ox家族基因启动子中的顺式作用元件预测分析 不同颜色方框分别代表不同顺式元件, 底部的比例尺表示启动子序列的长度。"

表3

2个花生品系农艺性状比较"

性状Trait P92 P92-1
株高 Plant height (cm) 36.59±1.11 38.33±1.82
分枝数 Branching number 10.33±0.47 9.67±0.47
结果数 Pod number 39.00±0.83 36.00±0.50
荚壳厚度 Pod shell thickness(mm) 1.15±0.06 1.48±0.22*
荚果长 Pod length (mm) 19.21±1.03 35.11±1.59**
荚果宽 Pod width (mm) 12.73±0.19 15.39±0.26**

图6

6个AhGA3ox家族基因在花生品系P92及P92-1各组织中表达模式分析 不同小写字母表示不同花生组织在0.05概率水平差异显著。"

图7

AhGA3ox家族基因在大小果花生品系果壳中表达模式分析 S1~S5分别表示果针进入土壤后第10、17、24、31和38天。*、**分别表示在0.05和0.01概率水平差异显著。"

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