基于CRISPR/Cas9技术创制耐盐香稻

doi:10.16819/j.1001-7216.2023.220907

中国水稻科学 ›› 2023, Vol. 37 ›› Issue (5): 478-485.DOI: 10.16819/j.1001-7216.2023.220907

基于CRISPR/Cas9技术创制耐盐香稻

李景芳¹, 温舒越², 赵利君², 陈庭木¹, 周振玲¹, 孙志广¹, 刘艳¹, 陈海元³, 张云辉³, 迟铭¹, 邢运高¹, 徐波¹, 徐大勇¹, 王宝祥¹^,^*()

¹连云港市农业科学院, 江苏连云港 222000
²南京农业大学, 南京 210095
³江苏省农业科学院种质资源与生物技术研究所, 南京 210014

收稿日期:2022-09-28 修回日期:2022-12-19 出版日期:2023-09-10 发布日期:2023-09-13
通讯作者: *email：wbxrice@163.com
基金资助:
连云港市财政专项资金资助项目(QNJJ2102);连云港市财政专项资金资助项目(QNJJ2211);连云港市财政专项资金资助项目(QNJJ1803);江苏省农业重大品种创制计划资助项目(PZCZ201704);江苏省政策引导类计划（苏北科技专项）资助项目(LYG-SZ202040)

Development of Aromatic Salt-tolerant Rice Based on CRISPR/Cas9 Technology

LI Jingfang¹, WEN Shuyue², ZHAO Lijun², CHEN Tingmu¹, ZHOU Zhenling¹, SUN Zhiguang¹, LIU Yan¹, CHEN Haiyuan³, ZHANG Yunhui³, CHI Ming¹, XING Yungao¹, XU Bo¹, XU Dayong¹, WANG Baoxiang¹^,^*()

¹Lianyungang Academy of Agricultural Science, Lianyungang 222000, China
²Nanjing Agricultural University, Nanjing 210095, China
³Institute of Germplasm Resources and Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

Received:2022-09-28 Revised:2022-12-19 Online:2023-09-10 Published:2023-09-13
Contact: *email：wbxrice@163.com

摘要/Abstract

摘要：

【目的】 为了促进耐盐香稻育种，利用CRISPR/Cas9系统对粳稻品种连粳11的Badh2和OsRR22基因进行编辑，以期快速获得一批不含有转基因成分且具有耐盐性和香味的纯合水稻材料。【方法】 根据Badh2和OsRR22基因序列中编辑位点的敲除效率设计靶位点，构建pH-Ubi-Cas9-Badh2-OsRR22敲除载体，利用农杆菌介导法转入受体品种连粳11中。对转基因后代进行潮霉素和Cas9标记PCR检测以及靶基因测序，获得无外源基因插入的badh2-osrr22纯合株系，并对后代种子性状和苗期耐盐性进行分析。【结果】 T₂代成熟种子中2-AP含量较背景材料连粳11显著增加，千粒重、粒长、粒宽无明显变化；128 mmol/L氯化钠处理14 d后株系21-30较连粳11苗高增加15.2%，苗鲜质量增加45.2%，苗干质量增加13.2%。【结论】 利用CRISPR/Cas9技术对Badh2和OsRR22基因编辑获得能稳定遗传且无外源基因插入的耐盐香稻材料，加快了水稻多性状聚合的选育进程。

关键词: 水稻, CRISPR/Cas9, 耐盐, 香味, Badh2, OsRR22

Abstract:

【Objective】 It is of importance to promote salt-tolerant and fragrant rice breeding. We obtained transgene free homozygous rice materials with salt-tolerance and fragrance by CRISPR/Cas9-mediated editing of Badh2 and OsRR22 in japonica variety Lianjing 11. 【Method】 Target sites were designed according to knockout efficiency of editing sites in Badh2 and OsRR22 gene sequence. The pH-Ubi-Badh2-OsRR22 knockout vector was constructed and transferred into the recipient variety Lianjing 11 by the Agrobacterium-mediated technology. PCR detection for hygromycin, Cas9 marker and target gene sequencing were performed in the transgenic progenies to obtain badh2-osrr22 homozygous lines without exogenous gene insertion. The seed traits and salt tolerance at seedling stage were analyzed. 【Result】 Compared with the background material Lianjing 11, the 2-AP content of T₂ significantly increased. After treated with 128 mmol/L NaCl solution for 14 days, the seedling height, the fresh and the dry weight of T₂ line 21-30 increased by 15.2%, 45.2% and 13.2%, respectively. 【Conclusion】 The successful editing of Badh2 and OsRR22 by CRISPR/Cas9 technology developed aromatic salt-tolerant homozygous lines without exogenous gene insertion, speeding up the breeding process of pyramiding multiple traits in rice.

Key words: rice, CRISPR/Cas9, salt-tolerant, fragrance, Badh2, OsRR22

李景芳, 温舒越, 赵利君, 陈庭木, 周振玲, 孙志广, 刘艳, 陈海元, 张云辉, 迟铭, 邢运高, 徐波, 徐大勇, 王宝祥. 基于CRISPR/Cas9技术创制耐盐香稻[J]. 中国水稻科学, 2023, 37(5): 478-485.

LI Jingfang, WEN Shuyue, ZHAO Lijun, CHEN Tingmu, ZHOU Zhenling, SUN Zhiguang, LIU Yan, CHEN Haiyuan, ZHANG Yunhui, CHI Ming, XING Yungao, XU Bo, XU Dayong, WANG Baoxiang. Development of Aromatic Salt-tolerant Rice Based on CRISPR/Cas9 Technology[J]. Chinese Journal OF Rice Science, 2023, 37(5): 478-485.

图/表 7

表1 本研究中使用的引物

Table 1. Primers used in this study.

引物名称 Primer name	引物序列 Primer sequence(5’-3’)
Badh2-S1-F	GGCATGGTGGAAAAAGTCCTATAG	构建Badh2敲除载体 Construction of Badh2 knockout vector
Badh2-S1-R	AAACCTATAGGACTTTTTCCACCA	构建Badh2敲除载体 Construction of Badh2 knockout vector
Badh2-F	CCTTTGTCATCACACCCTGG	Badh2敲除靶点测序鉴定
Badh2-R	TAACTGCCTTCCTTGCCACG	Sequencing for Badh2 knockout target
OsRR22-S1-F	GGCACTCAACTGACCATACAACTC	构建OsRR22敲除载体
OsRR22-S1-R	AAACGAGTTGTATGGTCAGTTGAG	Construction of OsRR22 knockout vector
OsRR22-F	TAGGAGGAAGTTCGGTAATCGT	OsRR22敲除靶点测序鉴定
OsRR22-R	ACATTTTCCCTGGTGAGTTTCT	Sequencing for OsRR22 knockout target
Cas9-F	CCTTCTGTGTGGTCTGGTTC	载体检测
Cas9-R	AGACAATCACCCCCTGGAAC	Vector assay
Hyg-F	CTATTTCTTTGCCCTCGGAC	转基因检测
Hyg-R	ATGCCTGAACTCACCGCGAC	Transgenic detection
qPCR SOS1-F	ATTGCAGCTGAGCATGTACG	实时荧光定量PCR
qPCR SOS1-R	AGAGCTTGCTTTCGTGTGAC	Quantitative real-time PCR
qPCR OsMYB2-F	GGGCTGAAACGCACAGGCAAGA	实时荧光定量PCR
qPCR OsMYB2-R	CTGCTTGGCGTGCTTCTGC	Quantitative real-time PCR
qPCR OsNHX1-F	GTGACAGACCTGGCAAATCC	实时荧光定量PCR
qPCR OsNHX1-R	TCGACACAGCTCCTCTCATC	Quantitative real-time PCR
qPCR OsHKT1;1-F	TGCCAGAAGTTGTTGAAGCC	实时荧光定量PCR
qPCR OsHKT1;1-R	CCCAGGAACATCACCAGGAT	Quantitative real-time PCR
qPCR OsP5CS1-F	GTCAGAGTGGACTGATGGCT	实时荧光定量PCR
qPCR OsP5CS1-R	GCCTTTCTAGTGCTGATGGC	Quantitative real-time PCR
UBIQUITIN-F	GCTCCGTGGCGGTATCAT	实时荧光定量PCR
UBIQUITIN-R	CGGCAGTTGACAGCCCTAG	Quantitative real-time PCR

图1 Badh2和OsRR22基因靶点和载体构建 A―Badh2和OsRR22基因结构及靶位点位置信息，绿色碱基代表PAM序列；B―Badh2和OsRR22基因靶点CRISPR/Cas9载体构建；C―表达载体中靶位点测序结果。

Fig. 1. Badh2 and OsRR22 target sites and CRISPR/Cas9 vector construction. A, Badh2 and OsRR22 structure and target site. The PAM motif is shown in green. B, Construction of CRISPR/Cas9 vector for Badh2 and OsRR22 gene target; C, Target site sequencing results in the expression vector.

图2 T1代部分植株Cas9标记鉴定结果 M―DNA标记；“+”―阳性对照；“−”―阴性对照；1~9为T1代部分植株。Cas9标记目标片段大小为906 bp。

Fig. 2. Identification of Cas9 markers in some T1 plants. M, DNA Marker; “+”, Positive control; “−”, Negative control; Lanes 1 to 9, Some plants of T1. The size of the Cas9 marker target fragment is 906 bp.

图3 T1代纯合株系Badh2和OsRR22突变类型红色箭头表示突变位置。“+”代表插入；“-”代表缺失。黑色箭头表示氨基酸位置。

Fig. 3. Mutation types at the Badh2 and OsRR22 loci of the homozygous lines in T1. Red arrows indicate mutation positions. “+” means insertion; “-” means deletion. Black arrows indicate amino acid positions.

图4 T2代两个纯合株系21-30、21-31苗期耐盐性鉴定数据用平均数±标准差表示。*和**分别表示在5%和1%水平上差异显著(t检验)。

Fig. 4. Salt tolerance identification of T2 homozygous lines 21-30, 21-31 at the seedling stage. Values are shown as mean ± SD. *, ** Significantly different at 0.05 and 0.01 levels by t-test, respectively.

图5 野生型连粳11与T2代21-30株系中耐盐相关基因的相对表达量数值用平均数±标准差表示。*表示在5%水平上差异显著(t检验)。

Fig. 5. Relative expression levels of salt resistance related genes in the wild-type and its T2 line 21-30. Values are shown as mean ± SD.*Significantly different at 0.05 level by t-test.

图6 野生型和转基因阳性株系中水稻籽粒和香味相关性状表现数值用平均数±标准差表示。**表示在1%水平上差异显著 (t 检验)。

Fig. 6. Performance of rice yield and fragrance related traits in the wild-type and its positive transgenic lines. Values are shown as mean ± SD.** Significantly different at 0.01 level by t-test.

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基于CRISPR/Cas9技术创制耐盐香稻

Development of Aromatic Salt-tolerant Rice Based on CRISPR/Cas9 Technology

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