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作物学报 ›› 2021, Vol. 47 ›› Issue (3): 385-393.doi: 10.3724/SP.J.1006.2021.04123

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

白菜型油菜srb多室性状的遗传分析与分子鉴定

杨阳(), 李淮琳(), 胡利民, 范楚川*(), 周永明   

  1. 华中农业大学作物遗传改良国家重点实验室, 湖北武汉 430070
  • 收稿日期:2020-06-05 接受日期:2020-09-13 出版日期:2021-03-12 网络出版日期:2020-10-09
  • 通讯作者: 范楚川
  • 作者简介:杨阳, E-mail: yangyangyy91@163.com;|李淮琳, E-mail: 1228023730@qq.com
  • 基金资助:
    国家自然科学基金项目资助(31671279);国家自然科学基金项目资助(31971976);国家自然科学基金项目资助(31371240)

Genetic analysis and molecular characterization of multilocular trait in the srb mutant of Brassica rapa L.

YANG Yang(), LI Huai-Lin(), HU Li-Min, FAN Chu-Chuan*(), ZHOU Yong-Ming   

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, Hubei, China
  • Received:2020-06-05 Accepted:2020-09-13 Published:2021-03-12 Published online:2020-10-09
  • Contact: FAN Chu-Chuan
  • Supported by:
    National Natural Science Foundation of China(31671279);National Natural Science Foundation of China(31971976);National Natural Science Foundation of China(31371240)

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摘要：

油菜多室角果是一种高产相关性状, 本研究对桑日白油菜(srb)多室性状的遗传调控机制进行研究。性状分析表明, 该突变体具有稳定的多室角果表型, 单株多室角果比例为94.7%~100.0%, 每角果平均3.5个心皮。遗传上srb突变体中的多室性状受1对隐性核基因控制。比较测序分析发现, srb中BrCLV3基因的CLE motif中存在一种新的单核苷酸突变(C/G), 可导致其保守结构域的第12位组氨酸突变为天冬氨酸, 将该位点命名为Brclv3Asp12。利用SNP标记进行分离群体的鉴定, 证实Brclv3Asp12中的C/G单核苷酸变异与多室表型共分离。转基因互补测验和体外多肽的处理试验进一步证实, 该材料中控制多室性状位点Brclv3Asp12突变导致了CLV3多肽活性的减弱, 是形成多室角果性状的原因。本研究初步阐明了白菜型油菜srb多室性状形成的机制。

关键词: 桑日白油菜, 多室角果, BrCLV3, 等位测验, 比较测序, 功能分析

Abstract:

Multilocular silique is considered as a trait associated with high yield in rapeseed, and we studied the genetic regulation of the multilocular trait in B. rapa var. srb. This mutant showed a stable multilocular phenotype, ranging from 94.7% to 100% multilocular siliques per plant and 3.5 carpels per silique. Genetic analyses showed that the multilocular trait was monogenically governed by a recessive nuclear gene. Comparative sequencing analysis revealed that there was a novel C-to-G single-nucleotide mutation in the core CLE motif of BrCLV3, leading to histidine mutation at position 12 in conserved domain to aspartic acid, which was named Brclv3Asp12. The analysis of segregated population by SNP marker showed that the C/G single-nucleotide variation in Brclv3Asp12 was co-segregated with the multilocular phenotype. Transgenic complementation studies and in vitro peptide assays further confirmed that the Brclv3Asp12 allelic mutation in srb could lead to reduced activity of the CLV3 peptide, resulting in the formation of multilocular phenotype. Therefore, the study preliminarily clarified the mechanism involved in multilocular silique formation in srb mutant.

Key words: B. rapa var. srb, multilocular silique, BrCLV3, allelism test, comparative sequencing, functional analysis

表1

本研究中所用的引物序列"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)
18S rRNA F AGGCCCGGGTAATCTTTG
18S rRNA R TCAACGCGAGCTGATGAC
DXP109 AAGGAGGTTTAAGGGATATGAGA
DXP113 TTAATTAAGGAGGTTTAAGGGATATGAGA
DXP114 GGCGCGCCTACAAAATGGATTCGAGGACTC
DXP117 TTAATTAACACGGGATGTTTAGAACACGGGA
DXP118 GTTGTTCTTTACTCTGGTAATGAGATAGGA
DXP120 TGTGACGCTTTAGGTAGTAGGCAG
DXP121 TACCAACGTCTTCATCGCCCAACT
DXP125 GCAAAGTGGTGACGTGAGCGAAAT
DXP129 GGGAGGAGCAAATGGAATTGAAG
DXP136 CCATGGATCTATTTAGGTACCCCAT
DXP246 AGGACCTGACCCTTTGCTCG
DXP247 CAGGACCTGACCCTTTGCATC
DXP248 AGGCTGCGAATTGTTGAATTTTT

图1

多室白菜型油菜的花器官和角果表型观察 a~b、c~f、g~j分别代表WT、多室ml4和srb的花器官数目比较; k~l、m~p、q~v分别代表WT、ml4和srb的角果及其横切面显示心皮数目变异; 横切石蜡切片显示在花发育第9~11时期的WT (两心皮, w)、ml4 (3~4心皮, x~y)和srb (2~4心皮, w~y)的雌蕊心皮变异; lc: locule; O: ovule; M: medial region; L: lateral region; 标尺为2 mm (k, m, o, q, s, u)、0.5 mm (l, o, p, r, t, v)和100 μm (w~y)。"

表2

亲本和F1材料的多室角果比例"

材料
Material		 范围
Range (%)		 均值±标准差
Mean±SD (%)
ml4 mutant 79.6-100.0 97.90±5.10
srb mutant 96.0-100.0 96.40±0.03
WT 0 —
ml4*srb F1 100.0 —
srb*ml4 F1 100.0 —
WT*srb F1 0 —
srb*WT F1 0 —

图2

BrCLV3基因结构和氨基酸序列比对 a: BrCLV3基因结构以及突变体srb与野生型材料间的碱基变异; 黑色方框表示编码区; 序列比较可以发现srb与野生型间存在2个单核苷酸突变和1个ATAT的插入/缺失突变。b: 白菜型油菜及拟南芥中CLV3同源基因的氨基酸序列比对; 箭头为CLV3信号肽序列剪切位点, 下划线为C端保守结构域CLE motif, 红色标识突变的氨基酸。"

图3

利用Brclv3Asp12等位基因特异性SNP标记对srb*WT的F2群体进行基因型和多室表型的共分离检测"

图4

转化35S::Brclv3Asp12可以部分恢复拟南芥clv3-2多室突变表型 野生型拟南芥ler为两室角果(a, b)和正常花序(q, r), clv3-2突变体的角果主要为5~6室(i~l)和异常增大的花序顶端分生组织(m, n), 35S::Brclv3Asp12转化clv3-2突变体产生了部分表型恢复单株, 其角果为2~6室(c~l)和较为正常花序(o, p)。部分恢复转基因家系中BrCLV3转录水平的RT-PCR检测(s)以及单株角果心皮数统计分析(t)。小写字母表示在0.05水平下差异显著。标尺为0.5 mm。"

图5

不同多肽处理对拟南芥SAM大小的影响 1: 未添加多肽(a, b)、添加1 μmol L-1 Brclv3Asp12多肽(c)和1 μmol L-1 BrCLV3多肽(d)处理下的野生型ler (a)和突变体clv3-2 (b, c, d)生长9 d的SAM观察; SAM相对大小是通过测量SAM侧平面上2个对生叶原基(箭头指示处)以上的面积; 标尺为50 μm。2: 不同多肽处理9 d后拟南芥SAM大小比较(e), 每种处理至少统计20个单株; 大写字母表示0.01水平下差异显著。"

图6

不同多肽处理对野生型拟南芥主根和RAM的影响 未添加多肽(a, h)和分别添加1 nmol L-1 (b, c, i, j, o, r)、10 nmol L-1 (d, e, k, l, p, s)、100 nmol L-1 (f, g, m, n, q, t)浓度的突变型多肽Brclv3Asp12 (b, d, f, i, k, m)和野生型多肽Brclv3 (c, e, g, j, l, n)处理8 d对拟南芥ler的主根生长的抑制作用。每种处理至少统计26个单株, **表示0.01水平下差异显著。a~g中标尺为1 cm, h~n为100 μm。黑色箭头指示根分生组织和伸长区的边界。"

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