基于热处理CRISPR/Cas9马铃薯植株的基因编辑体系优化

doi:10.16420/j.issn.0513-353x.2022-1168

摘要/Abstract

摘要：

以二倍体马铃薯栽培种AC142和四倍体栽培种E3为试验材料，构建并优化了马铃薯内源启动子驱动的CRISPR/Cas9基因编辑体系。以控制白化表型的八氢番茄红素脱氢酶基因（StPDS）为靶标基因，通过农杆菌介导的遗传转化侵染试管薯，根据转化效率和表型突变频率的分析，筛选出马铃薯内源启动子StU6和StUBI10驱动的HM4载体为最优CRISPR载体。以常温处理为对照，对HM4获得的AC142的37个全绿株系和白绿嵌合株系、E3的4个全绿株系的侧芽进行循环8次的短期热处理（37 ℃ 24 h + 22 ℃ 24 h）共15 d，表型分析和测序检测表明，对敲除株系进行短期热处理能显著提高CRISPR/Cas9的编辑效率。常温下，AC142和E3敲除株系的表型突变频率分别为16.2%（6/37）和0（0/4），循环8次短期热处理之后，表型突变频率分别提高到54.1%（20/37）和75%（3/4）。其中，AC142敲除株系的侧芽能得到10.8%（4/37）白化株系，而E3未能得到白化株系，只能得到75%（3/4）的白绿嵌合株系。4次连续15 d的短期热处理后，2.1%（3/144）的E3敲除株系的侧芽可在常温下保持白化。对短期热处理后的白化植株进行TA克隆，测序结果显示二倍体敲除株系CR-AC142-StPDS-17/21和四倍体敲除株系CR-E3-StPDS-3/4的8 ~ 12个单克隆都不存在野生型，说明本研究的敲除体系可在马铃薯中实现对靶标序列的完全诱导突变。该体系可广泛应用于马铃薯突变体的创制，为马铃薯基因功能组学研究和目标性状精准改良提供技术基础。

关键词: 马铃薯, 基因编辑, CRISPR/Cas9, StPDS, 热处理

Abstract:

The development of endogenous promoters driven CRISPR/Cas9 gene editing system in diploid potato cultivar AC142 and tetraploid cultivar E3 were reported. The StPDS gene was targeted by Agrobacterium-mediated potato microtuber transformation. Through the analysis of transformation efficiency and phenotypic mutation rate，HM4 driven by potato endogenous promoter StU6 and StUBI10 was selected as the optimal CRISPR vector. The short-term cycle of eight heat treatment（37 ℃ 24 h，22 ℃ 24 h）was performed on the lateral buds of 37 AC142 all-green lines and chimeric lines and four E3 all-green lines obtained from HM4 for 15 days. Phenotypic analysis and sequencing showed that short-term heat treatment of knockout lines could significantly improve the editing efficiency of CRISPR/Cas9. At room temperature，the phenotypic mutation rates of AC142 and E3 knockout lines were 16.2%（6/37）and 0（0/4），respectively. After one short-term heat treatment，the phenotypic mutation rates were increased to 54.1%（20/37）and 75%（3/4），respectively. Among them，AC142 knockout lines can obtain 10.8%（4/37） lines that maintain the albino phenotype，while E3 can only obtain 75%（3/4）chimeras with white spots. Four consecutive short-term heat treatments could result in 2.1%（3/144）albino mutants at room temperature in E3 knockout lines. TA cloning was performed on the albino lines after short-term heat treatment. The sequencing results showed that 8-12 single clones of the diploid knockout line CR-AC142-StPDS-17，21 and the tetraploid knockout line CR-E3-StPDS-3，four did not contain wild type，indicating that the knockout system in this study could induce mutations in the target sequence. The results provide a technical basis for functional genomics research and precise improvement of target traits in potato.

Key words: potatoes, gene editing, CRISPR/Cas9, StPDS, heat treatment

熊文汶, 刘慧敏, 陈凯园, 杜鹃, 宋波涛, 景晟林. 基于热处理CRISPR/Cas9马铃薯植株的基因编辑体系优化[J]. 园艺学报, 2023, 50(12): 2591-2600.

XIONG Wenwen, LIU Huimin, CHEN Kaiyuan, DU Juan, SONG Botao, JING Shenglin. Optimization of Gene Editing System Based on Heat-treated CRISPR/Cas9 Potato Plants[J]. Acta Horticulturae Sinica, 2023, 50(12): 2591-2600.

图/表 9

表1 引物序列

Table 1 Primer sequence

引物名称 Primer name	引物序列（5′-3′） Sequence of primers
pJCV55-F	AGCTCTCCCATATGGTCGACAAGGAATCTTTAAACATACG
pJCV55-R	CGGGAATTGGATCCGGTACCTCTAGATATCTCGAGTGCGG
pJCV-StUBI10-F	AGCTCTCCCATATGGTCGACCCCACCTTGAATGGATATGC
pJCV-StUBI10-Bsp-R	GCCTGCTTTTTTGTACACTTGAAAGAGAAATTTGAAGGC
GTR-StPDS-F	CGGGTCTCCTGCACAATCTTCTGGTCGTGGCATGTTTTAGAGCTAGAA
GTR-StPDS-R	TAGGTCTCTAAACAACCGATACTACTGGAGGCATGCACCAGCCGGGAA
Cas9-Check-F	GAGATACGACGAGCACCACC
Cas9-Check-R	CTTCACGGTCACTTTCCGGT
PDS-Check-F	TTGAGAGTCCAAGGTAGTTCAGCTT
PDS-Check-R	GTTTACGTAGCATCTCACAGAAAAGAC

图1 马铃薯StPDS基因两个靶标位点gRNA设计（A）及CRISPR敲除载体结构（B）示意图 HM1、HM2、HM3、HM4是载体名称，pRGEB32是HM1的载体骨架，pJCV55是HM2、HM3和HM4的载体骨架。

Fig. 1 gRNA design of two target sites of potato StPDS gene（A）and structure of CRISPR vector（B） HM1，HM2，HM3 and HM4 are carrier names，pRGEB32 is the carrier skeleton of HM1，and pJCV55 is the carrier skeleton of HM2，HM3 and HM4.

表2 马铃薯AC142和E3的CRISPR 载体转化效率和表型突变率的统计

Table 2 Statistics of CRISPR vector transformation efficiency and phenotypic mutation rate of potato AC142 and E3

载体名称 Vector name	转化受体 Transforma- tion receptor	外植体总数 Total number of explants	抗性芽总数 Total number of resistant buds	二次生根数 Number of secondary roots	转化效率/% Transforma- tion efficiency	表型Phenotype				表型突变频率/% Phenotypic mutation rate
载体名称 Vector name	转化受体 Transforma- tion receptor	外植体总数 Total number of explants	抗性芽总数 Total number of resistant buds	二次生根数 Number of secondary roots	转化效率/% Transforma- tion efficiency	白化 Albino	嵌合 Mosaic	全绿 No albino		表型突变频率/% Phenotypic mutation rate
HM1	E3	106	105	1	1	0	0		1	0
HM1	AC142	164	103	0	0	0	0		0	0
HM2	E3	149	159	2	1	0	0		2	0
HM2	AC142	216	188	2	1	0	0		2	0
HM3	E3	147	157	2	1	0	0		2	0
HM3	AC142	581	521	41	8	2	2		37	10
HM4	E3	157	206	4	2	0	0		4	0
HM4	AC142	299	461	44	10	4	6		34	23

图2 马铃薯AC142和E3野生型（WT）与其CRISPR敲除StPDS株系（CR）的表型图

Fig. 2 Phenotypic diagram of potato AC142 and E3 wild type（WT）and its CRISPR knockout StPDS lines（CR）

图3 马铃薯AC142和E3 的CRISPR敲除StPDS株系（CR）热处理白化图 A ~ B：于AC142中敲除StPDS得到的白绿嵌合株系和白化株系;C ~ E：于E3中敲除StPDS得到的嵌合株系、腋芽返绿株系和白化株系。HT→RT指1次热处理后将白化芽置于常温生长，2HT→RT和4HT→RT指连续2次和4次热处理后将白化的芽置于常温生长。红色箭头指热处理后白化的芽，将其单独取下置于常温生长，观察是否返绿。

Fig. 3 The heat treatment albino map of CRISPR knockout StPDS lines（CR）of potato AC142 and E3 A-B：the white green chimeric and albino lines obtained by knocking out StPDS in AC142;C-E：chimeric lines，axillary green returning lines，and albino lines obtained by knocking out StPDS in E3. HT→RT refers to the growth of albino buds at room temperature after one short-term heat treatments，2HT→RT and 4HT → RT refers to the growth of albino buds at room temperature after two and four consecutive short-term heat treatments. The red arrow refers to the albino bud after high temperature，which is taken down separately and grown at room temperature to observe whether it turns green.

表3 短期热处理对马铃薯AC142和E3的CRISPR敲除StPDS株系表型的影响

Table 3 Effect of short-term heat treatment on the phenotype of CRISPR knockout StPDS lines of potato AC142 and E3

马铃薯 Potato	阳性苗株数 Number of transgenic lines	处理 Treatment	表型Phenotype			突变频率 Frequency of induced mutants
马铃薯 Potato	阳性苗株数 Number of transgenic lines	处理 Treatment	白化 Albino	嵌合 Mosaic	全绿 No albino	突变频率 Frequency of induced mutants
AC142	37	22 ℃常温Room temperature	0	6	31	6/37
AC142	37	37 ℃热处理Heat treatment	4	16	17	20/37
E3	4	22 ℃常温Room temperature	0	0	4	0/4
E3	4	37 ℃热处理Heat treatment	0	3	1	3/4

图4 基因编辑株系靶位点的突变类型 RT：常温处理;HT：热处理。黑色加粗字为原间隔子相邻基序，蓝色字为靶向序列的gRNA，红色字为点突变位点，插入和缺失分别用+和-表示，靶序列左边括号中的数字分别代表相应处理和突变类型的单克隆数量，右边括号中的碱基数代表该克隆的插入和缺失的总碱基数。

Fig. 4 Mutation types of target sites in gene editing lines RT：Room temperature treatment;HT：Heat treatment. The black bold font indicates the protospacer-adjacent motif sequences，the blue font represents the gRNA of the target sequence，and the red font is the point mutation site. The insertion and deletion are represented by + and-，respectively. The numbers in the left bracket of the target sequence represent the number of monoclonals for the corresponding treatment and mutation types，respectively. The number of bases in the brackets on the right represents the total number of bases inserted and deleted in the clone.

表4 基因编辑株系的突变类型分析

Table 4 Mutation types of target sites in gene editing lines

温度/℃ Temperature	突变类型 Indel types	碱基数/bp Number of base	位点1（%）gRNA 1 sites（%）	位点2（%）gRNA 2 sites（%）
22	点突变，单碱基缺失或插入Point mutation，single base deletion or insertion	1	5（12.5）	8（20.0）
	小片段缺失Small fragment deletion	3 ~ 31	5（12.5）	11（27.5）
	大片段缺失或插入Long fragment deletion or insertion	—	—	—
37	点突变，单碱基缺失或插入Point mutation，single base deletion or insertion	1	17（35.4）	12（25.0）
	小片段缺失Small fragment deletion	2 ~ 59	15（31.3）	26（54.2）
	大片段缺失或插入Long fragment deletion or insertion	73 ~ 1434	7（14.6）	7（14.6）

图5 马铃薯E3的CR-E3-StPDS-4敲除株系中StPDS基因TA克隆的PCR检测结果 WT：野生型对照，1 ~ 19：CR-E3-StPDS-4敲除株系靶序列的19个单克隆。

Fig. 5 TA clone PCR detection results of CR-E3-StPDS-4 knockout lines WT is the E3 wild type control，1-19 refer to 19 monoclonals of the target sequence of the CR-E3-StPDS-4 knockout line.

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