CRISPR/Cas9技术编辑Wx基因创制新型糯稻种质  

Creating Novel Glutinous Rice Germplasms by Editing Wx Gene via CRISPR/Cas9 Technology

作　　者：黄李春 顾正文 谈红艳 赵微 肖颖[1] 储睿 范晓磊 张昌泉 李钱峰 刘巧泉[1,2] 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,Yangzhou 225009;Co-Innovation Center for Modern Production Technology of Grain Crops of Jiangsu Province/Key Laboratory of Crop Genetics and Physiology of Jiangsu Province,Yangzhou 225009)

机构地区：[1]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,扬州225009 [2]江苏省粮食作物现代产业技术协同创新中心/江苏省作物遗传生理重点实验室,扬州225009

出　　处：《植物遗传资源学报》2021年第3期789-799,共11页Journal of Plant Genetic Resources

基　　金：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)。

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。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 12 th and 13 th 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.

关 键 词：水稻 WX基因 GBSSI蛋白 直链淀粉含量 CRISPR/Cas9 

分 类 号：S511.23[农业科学—作物学]