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作物学报 ›› 2024, Vol. 50 ›› Issue (6): 1514-1524.doi: 10.3724/SP.J.1006.2024.34154

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

MeLAZY1c基因调控木薯株型的初步研究

望嘉翔1(), 郁雪婷1, 李梦桃1, 麦伟涛1, 陈新2,3,*(), 王文泉1,*()   

  1. 1海南大学热带农林学院 / 三亚南繁研究院, 海南三亚 572000
    2中国热带农业科学院热带生物技术研究所 / 海南热带农业资源研究院海南省热带农业生物资源保护与利用重点实验室, 海南海口 571101
    3中国热带农业科学院三亚研究院, 海南三亚 572000
  • 收稿日期:2023-09-12 接受日期:2024-01-12 出版日期:2024-06-12 网络出版日期:2024-02-09
  • 通讯作者: * 陈新, E-mail: chenxin@itbb.org.cn; 王文泉, E-mail: wangwenquan@itbb.org.cn
  • 作者简介:E-mail: 763601169@qq.com
  • 基金资助:
    国家自然科学基金NSFC-CG联合基金重点项目(3181101517);国家重点研发计划项目(2018YFD1000500);海南省高校研究生创新科研课题(Qhys2022-82)

Preliminary study on the regulation of cassava plant type by MeLAZY1c gene

WANG Jia-Xiang1(), YU Xue-Ting1, LI Meng-Tao1, MAI Wei-Tao1, CHEN Xin2,3,*(), WANG Wen-Quan1,*()   

  1. 1College of Tropical Agriculture and Forestry / Sanya Nanfan Research Institute, Hainan University, Sanya 572000, Hainan, China
    2Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences / Hainan Key Laboratory of Conservation and Utilization of Tropical Agricultural Biological Resources, Hainan Institute of Tropical Agricultural Resources, Haikou 571101, Hainan, China
    3Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya 572000, Hainan, China
  • Received:2023-09-12 Accepted:2024-01-12 Published:2024-06-12 Published online:2024-02-09
  • Contact: * E-mail: chenxin@itbb.org.cn;E-mail: wangwenquan@itbb.org.cn
  • Supported by:
    National Natural Science Foundation of China NSFC-CG Joint Fund Key Project(3181101517);National Key Research and Development Program of China(2018YFD1000500);Innovation Research Project for Graduate Students in Hainan Province(Qhys2022-82)

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摘要：

IGT基因家族参与作物株型的调控, LAZY属于IGT的亚家族。以拟南芥6个LAZY成员氨基酸序列为“种子”在木薯基因组中进行比对, 在木薯中共鉴定到8个LAZY基因, 其中MeLAZY1c与调控分枝角度的AtLAZY1高度同源。基于此, 本研究以MeLAZY1c为研究对象, 利用qRT-PCR分析发现MeLAZY1c在茎中转录水平最高, GUS染色显示pMeLAZY1c在维管束中染色较深。在MeLAZY1c启动子中发现8个光响应/调节元件, 随后发现黑暗能显著抑制其表达水平。同时对MeLAZY1c进行基因编辑, 获得纯合编辑株系19个, 炼苗移栽后观测表型, 发现melazy1c突变体植株与SC8野生型相比, 其主茎呈现匍匐生长, 并且弯曲部位茎外皮细胞形态扭曲变形且大小不一致, 近地侧1 mm处细胞数目约是远地侧细胞数量的1.5倍, 表明MeLAZY1c在木薯直立/匍匐生长建成方面发挥着重要作用。

关键词: 木薯, 株型调控, LAZY, 光响应, 匍匐, 基因编辑

Abstract:

The IGT gene family is involved in the regulation of crop plant type, and LAZY belongs to the IGT subfamily. By comparing the amino acid sequences of six Arabidopsis LAZY members as ‘seeds’ in the cassava genome, a total of 8 LAZY genes were identified in cassava, among which MeLAZY1c is highly homologous to AtLAZY1, which regulates branching angles. Based on this, MeLAZY1c had the highest transcription level in the stem by qRT-PCR and pMeLAZY1c was deeply stained in the vascular bundle by GUS staining, using MeLAZY1c as the experimental materials in this study. Eight photoresponsive/regulatory elements were found in the MeLAZY1c promoter, and we subsequently found that darkness significantly inhibited the relative expression level of MeLAZY1c. At the same time, gene editing of MeLAZY1c was performed and 19 homozygous edited lineages. The phenotype of MeLAZY1c mutants was observed after seedling transplantation, compared to the SC8 wild-type, the main stems of MeLAZY1c mutants had crawing growth, and the stem skin cells at the curved part were distorted and deformed with different sizes. The number of cells at 1 mm near the ground was about 1.5 times that of cells at the far ground. In conclusion, these results indicates that MeLAZY1c plays an important role in the establishment of upright/creeping growth of cassava.

Key words: cassava, plant type regulation, LAZY, light response, crawling, gene editing

表1

试验所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5°-3°)		 用途
Purpose
MeLAZY1c-cas9-F GATTATAGCAGAAGAGAATTCTGG 基因编辑
CRISPR-Cas9
MeLAZY1c-cas9-R AAACCCAGAATTCTCTTCTGCTAT
Tubulin-F GTGGAGGAACTGGTTCTGGA 实时荧光定量PCR
RT-qPCR
Tubulin-R TGCACTCATCTGCATTCTCC
MeLAZY1c-QPCR-F ATGCTGAAGAAAAAAATGTCCCA 实时荧光定量PCR
RT-qPCR
MeLAZY1c-QPCR-R TTCTTTTGGGGTTTATCTGCCTT
MeLAZY1c Hi-TOM-F ATAGAACATCCCTTGGCGAGC Hi-TOM 测序
Hi-TOM sequencing
MeLAZY1c Hi-TOM-R GCAGCAGAGCTCCTAGAAGAA
MeLAZY1c-Psl1p-F ACGCGTCGACATGAAGTTACTAGGTTGGATGC 亚细胞定位
Subcellular localization
MeLAZY1c-Psl1p-R CGGGATCCCAGCTCCAACACAAGGTAGT
MeLAZY1c-GUS-F AATTCGAGCTCGGTACCCGGGGATCCCCACAAGGAACATCAACTCAA GUS 载体连接
GUS carrier connection
MeLAZY1c-GUS-R CTCCTTTACTAGTCAGATCTACCATGGTTGACCAATAGCCTGTGTGATA

图1

LAZY家族进化树和MeLAZY表达水平 A: LAZY家族进化树; B: MeLAZY表达水平热图。"

图2

MeLAZY1c在不同品种组织的转录水平 不同小写字母代表显著性差异(P < 0.05)。"

图3

pMeLAZY1c转基因拟南芥不同组织的GUS染色 A, E: 子叶、腋芽和叶柄; B, F: 叶柄和下胚轴; C, G: 下胚轴; D, H: 根和根尖。A~D标尺为100 μm; E~F标尺为200 μm; G~H标尺为100 μm。"

图4

MeLAZY1c启动子顺式作用元件分析和不同时期光处理表达量检测 A: MeLAZY1c启动子顺式作用元件分布图; B: MeLAZY1c不同时期光处理表达量检测。不同小写字母代表显著性差异(P < 0.05)。"

图5

MeLAZY1c亚细胞定位 标尺为30 μm。"

图6

melazy1c突变体编辑类型及数量统计 A: Hi-Tom高通量测序检测melazy1c突变体编辑类型; B: 木薯melazy1c突变体编辑类型数量统计。"

图7

SC8与melazy1c突变体的表型比较 A: SC8与melazy1c突变体表型观察; B: melazy1c突变体和SC8的叶柄与主茎之间的角度; C: melazy1c突变体弯曲高度。*代表显著性差异(***, P < 0.001; ****, P < 0.0001)。"

图8

SC8和melazy1c突变体叶柄细胞形态观察"

图9

SC8和melazy1c突变体茎外皮细胞形态观察 A: SC8主茎横切面; B: melazy1c突变体主茎横切面; C: SC8主茎横切面放大图; D: melazy1c突变体主茎横切面放大图; E, G: SC8主茎近/远地侧纵切放大图; F, H: melazy1c突变体弯曲近/远地侧纵切放大图; I: SC8纵切; J: melazy1c突变体纵切; K~L: 1 mm近/远地侧细胞数量统计。"

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