引用本文
  • 黄李春,顾正文,谈红艳,等.CRISPR/Cas9技术编辑Wx基因创制新型糯稻种质[J].植物遗传资源学报,2021,22(3):789-799.    [点击复制]
  • HUANG Li-chun,GU Zheng-wen,TAN Hong-yan,et al.Creating Novel Glutinous Rice Germplasms by Editing Wx Gene via CRISPR/Cas9 Technology[J].植物遗传资源学报,2021,22(3):789-799.   [点击复制]
【打印本页】 【在线阅读全文】【下载PDF全文】 查看/发表评论下载PDF阅读器关闭

←前一篇|后一篇→

过刊浏览    高级检索

本文已被:浏览 392次   下载 365 本文二维码信息
码上扫一扫!
CRISPR/Cas9技术编辑Wx基因创制新型糯稻种质
黄李春, 顾正文, 谈红艳, 赵微, 肖颖, 储睿, 范晓磊, 张昌泉, 李钱峰, 刘巧泉
0
(扬州大学农学院植物功能基因组学教育部重点实验室/江苏省作物基因组学和分子育种重点实验室)
摘要:
控制水稻胚乳直链淀粉合成的 Wx 基因是决定稻米蒸煮食味品质的主效基因。适当降低稻米的直链淀粉含量可显著提高稻米蒸煮食味品质。编辑 Wx 基因编码的 GBSSI 蛋白 C 末端非关键位点有望微调其酶活性和胚乳直链淀粉含量,进而改良稻米蒸煮食味品质。在未检测到关键结构域和位点的 Wx 基因 3′端设计靶位点 T1 和 T2(分别位于 Wx 基因第 12 和 第 13 外显子),利用 CRISPR/Cas9 技术进行编辑。通过 PCR 和测序筛选获得纯合突变株系,用表观直链淀粉含量测定、凝胶渗透色谱、Western Blot 和 qRT-PCR 等技术分析其对水稻胚乳直链淀粉合成和 Wx 基因表达的影响。共获得 8 种保留 GBSSI主要结构域的纯合突变系 C1~C8。其中,C2 和 C3 中推测翻译的 GBSSI 蛋白仅有第 518~550/551 位密码子发生移码;C1、C4~C8 中推测翻译的 GBSSI 蛋白分别从第 551 位和第 517/518 位密码子开始移码。C1~C8 米粉中表观直链淀粉含量显著降低,从野生型的 16.79% 下降到 3.69%~4.44%,但均稍高于含自然突变 wx 基因的糯稻近等基因系的 2.92%。GPC 结果显示,突变系中无直链淀粉合成,但其支链淀粉中长链(Ap2)的链长均长于糯稻近等基因系 wx。qRT-PCR 和 Western Blot 结果显示,C1~C8 发育种子中 Wx 基因的转录水平无显著变化,但除 C2 和 C3 有一定量 GBSSI 蛋白表达外,其他纯合系中均无GBSSI 蛋白表达。综上所述,本研究通过 CRISPR/Cas9 技术创建了淀粉精细结构略区别于常规糯稻的新型糯稻种质 C1~C8,为水稻品质改良提供了新种质。同时也证明 GBSSI 蛋白 C 末端第 518~551 位氨基酸对其酶活性的形成有重要影响。这些结果为进一步解析 GBSSI 蛋白的结构以实现其精准编辑提供了参考。
关键词:  水稻  Wx 基因  GBSSI 蛋白  直链淀粉含量  CRISPR/Cas9
DOI:10.13430/j.cnki.jpgr.20210104003
投稿时间:2021-01-04修订日期:2021-02-03
基金项目:国家自然科学基金(31825019);国家转基因生物新品种培育重大专项(2016ZX08001006-005);江苏省重点研发计划(现代农业)项 目(BE2018357)
Creating Novel Glutinous Rice Germplasms by Editing Wx Gene via CRISPR/Cas9 Technology
HUANG Li-chun, GU Zheng-wen, TAN Hong-yan, ZHAO Wei, XIAO Ying, CHU Rui, FAN Xiao-lei, ZHANG Chang-quan, LI Qian-feng, LIU Qiao-quan
(Key Laboratory of Plant Functional Genomics of the Ministry of Education/Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, College of Agriculture, Yangzhou University)
Abstract:
The Wx gene,which controls the synthesis of amylose in rice endosperm,is the major gene that determines rice eating and cooking quality(ECQ). It can significantly improve rice ECQ by moderately reducing amylose content(AC)of endosperm. Editing the C-terminal of Wx encoded GBSSI enzyme to fine-tune its enzyme activity and grain AC is expected to further improve rice ECQ. By analyzing the functional domain of GBSSI through bioinformatics websites,two target sites,T1 and T2(located in the 12th and 13th exons of the Wx gene,respectively),were subjected for CRISPR/Cas9-medicated editing. The homozygous mutants were validated by PCR and Sanger sequencing,followed by quantifying the apparent amylose content,gel-permation chromatography,western blot and qRT-PCR. A total of 8 homozygous lines,C1-C8,with retained major domains of GBSSI were obtained. Among which,the 518-550/551 codons were shifted in the predicted protein in C2 and C3 lines and the predicted protein in C1,C4-C8 were shifted after codon 551,517 or 518. The apparent amylose content of C1-C8 was significantly reduced from 16.79% to 4.44%-3.69%,but significantly higher than that of near-isogenic line(NIL)carrying the conventional wx allele(AAC=2.92%). GPC results showed that there was no amylose synthesis in the selected mutants,but their chain length distribution of Ap2 was longer than that in NIL wx. The qRT-PCR and western blot suggested no significant difference on transcript of Wx gene but obvious reduction of GBSSI accumulation in the developing seeds in mutants. No GBSSI accumulation was detected in all the homozygous lines except for C2 and C3. As a result,this study generated glutinous rice lines with fine-tuned starch fine structure to conventional glutinous rice by CRISPR strategy,and provided evidence of the 518-551 amino acids on the formation of GBSSI enzyme activity. These results provide a reference for further analyzing the structure of GBSSI protein to achieve its precise editing.
Key words:  Oryza sativa L.  Wx gene  GBSSI  amylose content  CRISPR/Cas9

用微信扫一扫

用微信扫一扫