利用CRISPR/Cas9技术对水稻香味品质进行遗传改良

doi:10.7668/hbnxb.20190361

摘要/Abstract

摘要： 为了获得经济价值更高的香稻品种，利用成熟的CRISPR/Cas9技术，在水稻Badh2基因上设计sgRNA-E2和sgRNA-E3 2个靶位点，对超级稻品种龙粳31的香味品质进行改良，其中靶位点sgRNA-E2跨第2个内含子和第2个外显子，靶位点sgRNA-E3位于第3外显子区。经检测共获得有碱基突变的转基因植株6株，其中2个单株为纯合突变，4个单株为双等位突变。突变类型包括碱基缺失、插入及碱基变异，其中突变单株Badh2-E2-13有大片段缺失，为双等位突变，分别缺失64，108 bp。在转录水平上发现6个突变株系的Badh2基因表达量均比野生型显著降低（P<0.05）。通过气相色谱-质谱联用（GC-MS）技术检测T₀突变体种子的香味物质2AP含量，发现5个突变单株的2AP含量相对野生型显著提高（P<0.05），2个突变单株达到阳性对照（信香粳1号）水平，其中1个相比阳性对照显著提高。通过对6个突变株系的T₁主要农艺性状的调查发现，3个株系在香味物质含量提高的同时主要农艺性状和野生型没有显著差异。进一步对T₁植株进行筛选，共获得了无转基因序列的突变单株10个。综上，利用CRISPR/Cas9技术成功对超级稻品种龙粳31的香味品质进行了遗传改良，实现了香味物质含量的显著提高，为香稻和超级稻的育种提供了基础材料。

关键词: 香稻, 龙粳31, CRISPR/Cas9, 2-乙酰-1-吡咯啉, Badh2

Abstract: In order to obtain the fragrant rice varieties with higher economic value, two target sites were designed on Badh2 gene using CRISPR/Cas9 technology to improve the fragrance quality of super rice Longjing 31, the sgRNA-E2 between intron 2 and exon 2, and the sgRNA-E3 on exon 3 of Badh2 gene, respectively. Six transgenic lines were detected with base deletion, insertion and mutation, among which, four lines were di-allelic mutation and two lines were homozygous mutation. Large fragment deletion occurring in the di-allelic mutation Badh2-E2-13 with lack of 64,108 bp.qRT-PCR results indicated that companed with the wild type the transcriptional level of Badh2 declined significantly (P<0.05) in the six mutants. The fragrance content of 2AP increased dramatically (P<0.05) in the five mutations compared with wild type using gas chromatography-mass spectrometry (GC-MS) method, two of them achieved positive control (Xinxiangjing 1) level, in which one was significantly improved. The main agronomic traits of the T₁ of the 6 mutant lines were investigated, indicating that there was no significant difference between the wild type and the 3 lines, which of the fragrance content increased. Further, screening the marker gene of T₁ lines, 10 transgenic-free individuals were obtained. Comprehensively,the Badh2 gene was successfully edited by CRISPR/Cas9 technology in Longjing 31, increasing 2AP content of Longjing 31 remarkably, which would provide suitable material for fragrant and super rice breeding.

Key words: Fragrant rice, Longjing 31, CRISPR/Cas9, 2-acetyl-1-pyrroline, Badh2

中图分类号:

Q78
S511.03

周俊飞, 高利芬, 汪伟航, 陈利红, 方治伟, 李论, 李甜甜, 彭海. 利用CRISPR/Cas9技术对水稻香味品质进行遗传改良[J]. 华北农学报, 2020, 35(2): 57-64. doi: 10.7668/hbnxb.20190361.

ZHOU Junfei, GAO Lifen, WANG Weihang, CHEN Lihong, FANG Zhiwei, LI Lun, LI Tiantian, PENG Hai. Genetic Improvement of Fragrance Quality in Rice Using CRISPR/Cas9 Technology[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2020, 35(2): 57-64. doi: 10.7668/hbnxb.20190361.

http://www.hbnxb.net/CN/Y2020/V35/I2/57

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