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作物学报 ›› 2021, Vol. 47 ›› Issue (10): 1854-1862.doi: 10.3724/SP.J.1006.2021.04208

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

棉花花器官突变体的鉴定及候选基因的克隆

杨琴莉1(), 杨多凤1, 丁林云1, 赵汀2, 张军2, 梅欢2, 黄楚珺2, 高阳1, 叶莉1, 高梦涛1, 严孙艺2, 张天真1,2, 胡艳1,2,*()   

  1. 1南京农业大学作物遗传与种质创新国家重点实验室, 江苏南京 210095
    2浙江大学农业与生物技术学院, 浙江杭州 310058
  • 收稿日期:2020-09-10 接受日期:2021-01-13 出版日期:2021-10-12 网络出版日期:2021-03-11
  • 通讯作者: 胡艳
  • 作者简介:E-mail: 739768392@qq.com
  • 基金资助:
    国家自然科学基金项目(31970320);国家转基因生物新品种培育重大专项(2016ZX08009-003)

Identification of a cotton flower organ mutant 182-9 and cloning of candidate genes

YANG Qin-Li1(), YANG Duo-Feng1, DING Lin-Yun1, ZHANG Ting2, ZHANG Jun2, MEI Huan2, HUANG Chu-Jun2, GAO Yang1, YE Li1, GAO Meng-Tao1, YAN Sun-Yi2, ZHANG Tian-Zhen1,2, HU Yan1,2,*()   

  1. 1State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
    2College of Agricultural & Biotechnology, Zhejiang University, Hangzhou 310000, Zhejiang, China
  • Received:2020-09-10 Accepted:2021-01-13 Published:2021-10-12 Published online:2021-03-11
  • Contact: HU Yan
  • Supported by:
    National Natural Science Foundation of China(31970320);National Major Project for Developing New GM Crops(2016ZX08009-003)

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摘要：

棉花是世界性的重要经济作物, 是天然纤维的主要来源。棉花生殖生长过程现蕾、开花、结铃都直接影响棉花主要经济性状——棉纤维的产量和品质。本研究在转基因棉花材料中发现了1个花器官突变体, 命名为182-9, 其花器官呈现瓣化特征。PCR和Southern杂交证明突变体182-9中的外源基因已整合到基因组中, 且为单拷贝插入。通过基因组重测序进行序列比较发现, 突变体182-9基因组中外源T-DNA插入位点为染色体A11:59086840。PCR和Southern杂交对插入位点进行了进一步验证。根据棉花基因组注释结果, 在基因组插入位点附近有3个候选基因(GH_A11G2251、GH_A11G2252和GH_A11G2253)。其中GH_A11G2251为AP2类基因。已有研究证明, AP2类基因为花器官ABC模型中控制萼片和花瓣形成的A类功能基因。qRT-PCR结果显示, GH_A11G2251在转基因受体W0的花瓣、雌蕊和雄蕊3个组织中的表达与突变体182-9中存在显著性差异。本研究为进一步深入探究棉花花器官发育的分子机制研究提供了参考。

关键词: 棉花, 转基因, 花器官, 突变体, 基因克隆

Abstract:

Cotton is an important economic crop and the main source of natural fiber in the world. The budding, flowering and bolling during cotton growth and development directly affect the yield and quality of cotton fiber that are the main economic traits of cotton. In this study, we found a flower organ mutant (named 182-9) in transgenic cottons, which displayed the floral organ petaloid feature. PCR and Southern blotting confirmed that the foreign T-DNA was integrated into the 182-9 genome with a single copy. Comparative analysis of the resequencing data revealed that the exogenic T-DNA was inserted in the 182-9 on chromosome A11: 59086840. The insertion site was further verified by PCR and southern blot. According to the gene annotation of cotton genome, there were three candidate genes of GH_A11G2251, GH_A11G2252, and GH_ A11G2253, near to the insertion site. GH_ A11G2251 encoded AP2 genes controlling the formation of sepals and petals in the ABC model of flower organs as previous report. qRT-PCR showed that there were significant differences in the expression level of GH_A11G2251 in petals, pistil and stamens of transgenic receptor W0 and mutant 182-9. Our study provided the basis for further study of molecular mechanism in cotton floral organ development.

Key words: cotton, transgenic, floral organ, mutant, gene cloning

图1

转基因载体T-DNA序列"

图2

突变体182-9与转基因受体W0花器官表型比较 A: 转基因受体W0花器官整体结构与子房。B: 突变体182-9花器官结构。C: 转基因受体W0苞叶、萼片、花瓣、雌蕊雄蕊结构。D: 突变体182-9苞叶、萼片以及瓣化结构。标尺为1 cm。"

图3

PCR检测转基因材料(182-9) NPTII基因 M: DNA分子量Marker; W0: 阴性对照; 1: 转基因株系182-9; 2: 转基因株系182-36; 3: 转基因株系182-91; 4: 转基因株系182-150; 5: 转基因株系182-172; 6: 转基因株系182-173; 7: 转基因株系182-187。"

图4

Southern杂交检测突变体182-9中外源基因NPTII的整合 M: DNA marker; 1: 转基因受体W0; C: 质粒; 2: 转基因株系182-187; 3: 转基因株系182-36; 4: 转基因株系182-173; 5: 转基因株系182-9 (箭头所示)。"

表1

重测序数据评估"

样品
Sample		 原始reads
Raw reads		 过滤后reads
Clean reads		 原始碱基数
Raw base (Gb)		 过滤后碱基数Clean base (Gb) Q30
(%)		 GC含量
GC content (%)
182-9 245,670,776 245,536,479 73.70 73.66 91.81 35.43
W0 236,081,132 235,926,310 70.82 70.78 89.16 35.51

图5

PCR检测外源T-DNA在基因组中插入位点 A: PCR扩增条带; B: 扩增序列的测序结果(红色部分为棉花基因组序列, 其他为T-DNA序列)。M: DNA marker; W0: 转基因受体; 182-9: 转基因株系。"

图6

插入位点的Southern检测 M: DNA marker; C: 阳性质粒; 1: 转基因株系182-9; 2: 转基因受体W0。箭头所示为转基因182-9中特异性条带。"

表2

候选基因的注释信息"

基因ID
Gene ID		 拟南芥中同源基因
Homologous genes in Arabidopsis		 功能描述
Function annotation
GH_A11G2251 ANT AP2类乙烯反应转录因子
AP2-like ethylene-responsive transcription factor ANT
GH_A11G2252 At1g65750 假定核糖核酸酶H蛋白At1g65750
Putative ribonuclease H protein At1g65750
GH_A11G2253 NA NA

图7

候选基因与插入位点的相对位置"

图8

GH_A11G2251在182-9和W0两个材料中的扩增序列比较"

图9

GH_A11G2252在182-9和W0两个材料中的扩增序列比较"

图10

GH_A11G2253在182-9和W0两个材料中的扩增序列比较"

图11

候选基因的在花器官中表达分析 A: W0和182-9组织示意图; B: 候选基因在突变体182-9及其野生型W0中的表达水平。其中, *表示在0.05水平差异显著。"

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