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作物学报 ›› 2023, Vol. 49 ›› Issue (2): 354-364.doi: 10.3724/SP.J.1006.2023.22002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

利用CRISPR/Cas9探究水稻OsPIN5c基因功能

杨晓祎1(), 王慧慧1, 张艳雯1, 侯典云1, 张红晓1, 康国章2, 胥华伟1,*()   

  1. 1河南科技大学农学院, 河南洛阳 471000
    2河南农业大学省部共建小麦玉米作物学国家重点实验室, 河南郑州 450046
  • 收稿日期:2022-01-13 接受日期:2022-06-07 出版日期:2022-07-08 网络出版日期:2022-07-08
  • 通讯作者: 胥华伟
  • 作者简介:E-mail: 1961827259@qq.com
  • 基金资助:
    河南省自然科学基金项目(182300410012);河南省自然科学基金项目(202300410151);河南省自然科学基金项目(202300410340);小麦玉米作物学国家重点实验室开放课题项目(SKL2021KF03)

Function analysis of OsPIN5c gene by CRISPR/Cas9

YANG Xiao-Yi1(), WANG Hui-Hui1, ZHANG Yan-Wen1, HOU Dian-Yun1, ZHANG Hong-Xiao1, KANG Guo-Zhang2, XU Hua-Wei1,*()   

  1. 1College of Agriculture, Henan University of Science and Technology, Luoyang 471000, Henan, China
    2National Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou 450046, Henan, China
  • Received:2022-01-13 Accepted:2022-06-07 Published:2022-07-08 Published online:2022-07-08
  • Contact: XU Hua-Wei
  • Supported by:
    Natural Science Foundation of Henan Province(182300410012);Natural Science Foundation of Henan Province(202300410151);Natural Science Foundation of Henan Province(202300410340);Open Research Fund of National Key Laboratory of Wheat and Maize Crop Science(SKL2021KF03)

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摘要：

生长素极性运输在植物生长发育中发挥着重要作用, 生长素输出载体蛋白(PIN-FORMED, PIN)是控制生长素极性运输的关键蛋白。虽然部分水稻OsPIN基因功能已有报道, 但水稻OsPIN5c的生物学功能仍有待研究。本研究在OsPIN5c第1外显子处设计靶点并构建CRISPR/Cas9载体, 通过转化粳稻品种日本晴获得24株转基因植株, PCR产物直接测序分析表明其中15株发生突变, 突变率为62.5%, 突变类型为双等位突变; 进一步从T1代突变体中筛选获得3种不同的纯合突变体, 将其命名为ospin5c-1、ospin5c-2和ospin5c-3。序列比对分析表明, 这3种类型的突变均造成移码突变和蛋白翻译提前终止, 由原来的398个氨基酸分别缩短为109、106和250个氨基酸; 跨膜螺旋结构域分析表明, 3种突变体中OsPIN5c蛋白的跨膜结构完全消失; 蛋白结构预测表明3种突变体中OsPIN5c蛋白螺旋结构明显减少。突变体的苗高、根长和不定根数均显著低于野生型植株。组织特异性表达表明OsPIN5c主要在根中表达。突变体根中OsPIN1a和OsPIN5b表达量提高; 生长素合成关键基因OsYUC1、OsYUC4、OsYUC6和OsYUC7的表达量也显著提高。ospin5c突变体根的向重性受到部分抑制。本研究利用CRISPR/Cas9基因编辑技术获得ospin5c纯合突变体并探讨了OsPIN5c基因功能, 为利用OsPIN5c基因进行作物遗传改良提供了潜在的基因资源。

关键词: OsPIN5c, CRISPR/Cas9, 基因功能, 水稻

Abstract:

Polar auxin transport (PAT) plays a key role in plant growth and development, and auxin efflux carriers PIN-FORMED (PIN) are the crucial proteins controlling PAT. Although the functions of some OsPIN genes have been reported, the function of OsPIN5c gene is still unclear. In this study, the target site was designed at the first exon of OsPIN5c and the recombinant CRISPR/Cas9 vector of OsPIN5c was constructed. Twenty-four independent transgenic rice lines were obtained by transformation using Nipponbare (Oryza sativa L. ssp. japonica) as the materials. PCR product sequencing indicated that 15 lines were identified as mutants and the corresponding mutation rate was 62.5%, the mutation type were biallelic heterozygous mutations. Three independent ospin5c homozygous mutants were further obtained in T1 generation lines and named as ospin5c-1, ospin5c-2, and ospin5c-3, respectively. Sequence alignment analysis showed that the three types of mutations resulted in frame-shift mutation and premature translation termination, which were shortened from 398 amino acid (aa) in wild-type (WT) plants to 109 aa, 106 aa and 250 aa, respectively. Transmembrane helices (TMH) indicated that the TMH of these three mutation proteins were disappeared totally. Protein structure demonstrated that the helix of three mutation proteins were obviously reduced than the native OsPIN5c protein. Phenotype structure indicated that, compared to the WT, the shoot height, root length and adventitious root number were significantly decreased in ospin5c mutants at seedling stage. Tissue-specific analysis showed that OsPIN5c was highly expressed in roots, and OsPIN genes (OsPIN1a and OsPIN5b), OsYUC genes (OsYUC1, OsYUC4, OsYUC6, and OsYUC7) were up-regulated significantly in ospin5c mutants. The gravitropism response of ospin5c mutants was partially inhibited. In conclusion, three ospin5c homozygous mutants were obtained via CRISPR/Cas9 technology and the function of OsPIN5c was investigated in this study, providing the potential gene resources for crop genetic improvement by using OsPIN5c gene.

Key words: OsPIN5c, CRISPR/cas9, gene function, rice

表1

引物及序列"

引物名称Primer name 正向引物Forward sequence (5'-3') 反向引物Reverse sequence (5'-3')
OsPIN5c-CRISPR TGTGTGGGTTCTCGTGGTGCATCAC AAACGTGATGCACCACGAGAACCCA
HPT CTGAACTCACCGCGACGTCTGTC TAGCGCGTCTGCTGCTCCATACA
OsPIN5c-Assay CCTCGTCGCCTGCTTCGCCG CCACACCGCTCTCACCAGCGG
OsPIN1a-qPCR CCTGAAATCCATCTCCATCCTC AACGTCGCCACCTTGTT
OsPIN5b-qPCR GCAAAGGAGTATGGGCTTCA GCAATCAGAATCGGCAGAGA
OsYUC1-qPCR AGGTGTTGGTCGTGGGATGCG GCGATGCCGAACGTGGATAGA
OsYUC4-qPCR CCTCGACCTCTGCAACCACAATG CGACAACAGGAGTACCAGCCAATC
OsYUC6-qPCR GGATACCAAAGCAACGTCCCC TGAAGCCAACAGAGTAGAGCCCTG
OsYUC7-qPCR ACCGGCTACCGCAGCAATGTG CGTACAGCCCCGACTCACCCT
OsACTIN1-qPCR CTTCATAGGAATGGAAGCTGCG CACCTTGATCTTCATGCTGCTA

图1

OsPIN5c gRNA靶位点示意图及阳性克隆筛选 A: OsPIN5c靶位点示意图; PAM: 前间隔序列邻近基序; 黑色下画线表示PAM序列。B: PCR筛选阳性克隆, M: DL2000 DNA marker; 1~12: PCR产物。"

图2

T0代突变植株的筛选及分析 A: PCR扩增OsPIN5c基因组片段; M: DL2000 DNA marker; 1~7: PCR产物。B: 突变类型分析, PAM: 前间隔序列邻近基序; 黑色下画线表示PAM序列。"

图3

T1代纯合ospin5c突变体的筛选 PAM: 前间隔序列邻近基序; 黑色下画线表示PAM序列; 红色箭头表示突变位点。"

图4

无转基因成分植株筛选 A: PCR筛选结果; M: DL2000 DNA marker; 1~25: HPT PCR扩增结果; P: 阳性对照(质粒); N: 阴性对照(WT)。B: 叶片潮霉素抗性筛选结果, 1~25: ospin5c离体叶片潮霉素筛选。"

图5

OsPIN5c突变蛋白的氨基酸变化 A: 靶位点氨基酸变化; PAM: 前间隔序列邻近基序; 下画线对应的氨基酸表示突变氨基酸; 星号表示翻译的终止。B: 突变蛋白比对分析。"

图6

OsPIN5c突变蛋白的跨膜螺旋结构域及三级结构分析 A: 跨膜螺旋结构域分析; B: 三级结构分析。"

图7

幼苗期ospin5c突变体表型 A: 7 d ospin5c突变体表型分析。B: 14 d ospin5c突变体表型分析(n ≥ 15)。**表示P < 0.01水平差异。"

图8

基因表达分析 A: OsPIN5c组织特异性表达分析。B: OsPIN和OsYUC表达分析; *表示P < 0.05水平差异, **表示P < 0.01水平差异, ***表示P < 0.001水平差异。"

图9

野生型(WT)和ospin5c突变体根向重性分析 A: 重力刺激后野生型(WT)和ospin5c突变体表型, 0~24 h表示重力刺激处理时间。B: 重力刺激后野生型(WT)和ospin5c突变体根弯曲角度统计分析(n ≥ 12); *表示P < 0.05水平差异, **表示P < 0.01水平差异。"

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