利用真空渗透和CRISPR/Cas9系统获得非转基因菜薹突变体

doi:10.13560/j.cnki.biotech.bull.1985.2022-0042

摘要/Abstract

摘要：

通过CRISPR/Cas9基因编辑系统获得非转基因菜薹突变体，实现CRISPR/Cas9体系在菜薹原位转化中的应用，为无选择标记的基因编辑技术应用提供参考。以‘49菜心’为试材，番茄红素脱氢酶基因（PDS）为靶基因，采用真空渗透原位转化方法，转化菜薹25株。结果表明，在2 032株原位转化种子苗中鉴定出3株发生PDS基因编辑，其中1株有外源转化载体插入，PDS发生杂合编辑，表型与野生型一致。另外2株具有矮化和失绿表型，且基因组无外源载体片段插入，PDS发生不同类型的敲除突变。CRISPR/Cas9通过不依赖组织培养的原位转化可以在菜薹中实现基因编辑，并能直接获得无外源插入的非转基因编辑突变体。

关键词: 菜薹, 无筛选标记, 真空渗透转化, 不依赖组织培养, CRISPR/Cas9

Abstract:

The objective is to acquire non-transgenic Brassica campestris mutants via CRISPR/Cas9 gene editing system，for achieving the application of CRISPR/Cas9 in B. campestris in planta transformation method and providing reference for application of marker-free gene editing technique. Using ‘49 Caixin’ as plant material and dehydrogenase gene（PDS）as target gene，total of 25 plants were transformed by in planta transformation via vacuum-infiltration method. The results showed that 3 of the 2 032 transformed seedlings were identified to have PDS gene editing，one of which was detected to have exogenous vector insertion and heterozygous editing of PDS gene，and the phenotype was consistent with that of the wild type. The other two seedlings had a dwarf，albino phenotype，and there was no insertion of foreign vector in their genomes. Different types of targeted mutagenesis of PDS gene were detected in these two seedlings compared with the wild type. In conclusion，gene editing can be achieved in B. campestris by CRISPR/Cas9 through in planta tissue culture-independent transformation，and particularly non-transgenic editing mutants without exogenous insertion can be obtained directly.

Key words: Brassica campestris, marker-free, vacuum infiltration transformation, tissue culture-independent, CRISPR/Cas9

宗梅, 韩硕, 郭宁, 段蒙蒙, 刘凡, 王桂香. 利用真空渗透和CRISPR/Cas9系统获得非转基因菜薹突变体[J]. 生物技术通报, 2022, 38(10): 159-163.

ZONG Mei, HAN Shuo, GUO Ning, DUAN Meng-meng, LIU Fan, WANG Gui-xiang. Production of Marker-free Mutants of Brassica campestris Mediated by CRISPR/Cas9 Through Vacuum Infiltration[J]. Biotechnology Bulletin, 2022, 38(10): 159-163.

图/表 2

表1 本研究使用的引物

Table 1 Primers used in this study

引物名称 Primer name	序列 Sequence（5'-3'）	目的 Purpose
nCas9-IDF	CATACCTCCCAGAACACAAATAAGC	扩增nCas9片段
nCas9-IDR	ACTGAAGGGCAATAGTGAAGAATGT	扩增nCas9片段
gRNA-IDF	TGTCCCAGGATTAGAATGATTAGGC	扩增gRNA片段
RNA-IDR	CCCCAGAAATTGAACGCCGAAGAAC	扩增gRNA片段
PDS-1	ATGCAACTGATCAATGCGGT	扩增含有靶点的PDS基因片段
PDS-356	CGGGAATATCGCAGCTAAAG	扩增含有靶点的PDS基因片段
PDS-F	ATTGAACGAGAAGAAGCAAGCCT	载体构建
PDS-R	AAACAGGCTTGCTTCTTCTCGTT	载体构建

图1 利用CRISPR/Cas9系统通过真空渗透原位转化获得菜薹PDS突变体（a）：‘49菜心’PDS基因1-4个外显子的DNA序列，以不同背景颜色显示不同的外显子。第一外显子末端的编辑靶点用下划线标注，PAM区域CCC（互补序列为GGG）用红色字体表示；（b）：利用pBSE401载体进行真空渗透转化，靶点的19个碱基插入到sgRNA区，载体携带Bar基因筛选标记；（c）：Bar试纸呈阳性的#1植株PDS基因的PCR测序结果，显示在靶点区发生多峰变异；（d）：2 032株幼苗中有2株（#2和#3）表现矮小白化，与PDS基因突变体的表型一致，图示为#2植株；（e）：#2和#3植株的测序结果表明，两个植株的PDS基因在靶点区与野生型相比均发生了多种类型的突变

Fig. 1 CRISPR/Cas9-mediated gene editing and vacuum infiltration in planta transformation to creates PDS mutants in B. campestris （a）：DNA sequence of the first four exons（highlighted in different colors）of PDS of ‘49 Caixin’. The sgRNA at the end of the first exon is underlined and the PAM region CCC（complementary sequence is GGG）is shown in red letter.（b）：pBSE401 vector was used in vacuum infiltration transformation and the transcribed sgRNA was designed to have 19-base matches with the target. The vector carries Bar selection marker.（c）：Only plant #1 showed positive in Bar strip detection. And PDS gene sequencing of plant #1 showed sequence variation in the target region.（d）：Two of the 2 032 seedlings（plant #2 and plant #3）were weak albino，which was consistent with the phenotypes of PDS mutants. The picture shows plant #2.（e）：Sanger sequencing of plant #2 and plant #3 revealed the targeted mutagenesis of PDS gene in 2 plants compared with the wild type

参考文献 20

[1]	Chen LZ, Li W, Katin-Grazzini L, et al. A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants[J]. Hortic Res, 2018, 5:13. doi: 10.1038/s41438-018-0023-4 URL
[2]	Iaffaldano B, Zhang YX, Cornish K. CRISPR/Cas9 genome editing of rubber producing dandelion Taraxacum kok-saghyz using Agrobacterium rhizogenes without selection[J]. Ind Crops Prod, 2016, 89:356-362. doi: 10.1016/j.indcrop.2016.05.029 URL
[3]	Woo JW, Kim J, Kwon SI, et al. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins[J]. Nat Biotechnol, 2015, 33(11):1162-1164. doi: 10.1038/nbt.3389 pmid: 26479191
[4]	Murovec J, Guček K, Bohanec B, et al. DNA-free genome editing of Brassica oleracea and B. rapa protoplasts using CRISPR-Cas9 ribonucleoprotein complexes[J]. Front Plant Sci, 2018, 9:1594. doi: 10.3389/fpls.2018.01594 pmid: 30455712
[5]	Park J, Choe S. DNA-free genome editing with preassembled CRISPR/Cas9 ribonucleoproteins in plants[J]. Transgenic Res, 2019, 28(Suppl 2):61-64. doi: 10.1007/s11248-019-00136-3 pmid: 31321685
[6]	Gerszberg A. Tissue culture and genetic transformation of cabbage(Brassica oleracea var. capitata):an overview[J]. Planta, 2018, 248(5):1037-1048. doi: 10.1007/s00425-018-2961-3 pmid: 30066219
[7]	Qing CM, Fan L, Lei Y, et al. Transformation of pakchoi(Brassica rapa L. ssp. chinensis)by Agrobacterium infiltration[J]. Mol Breed, 2000, 6(1):67-72. doi: 10.1023/A:1009658128964 URL
[8]	侯喜林, 李英, 黄菲艺. 不结球白菜(Brassica campestris ssp. chinensis)主要性状及育种技术的分子生物学研究新进展[J]. 园艺学报, 2020, 47(9):1663-1677.
	Hou XL, Li Y, Huang FY. New advances in molecular biology of main characters and breeding technology in non heading Chinese cabbage(Brassica campestris ssp. chinensis)[J]. Acta Hortic Sin, 2020, 47(9):1663-1677.
[9]	Sivanandhan G, Moon J, Sung C, et al. L-cysteine increases the transformation efficiency of Chinese cabbage(Brassica rapa ssp. pekinensis)[J]. Front Plant Sci, 2021, 12:767140. doi: 10.3389/fpls.2021.767140 URL
[10]	张广辉, 巩振辉, 薛万新, 等. 大白菜和油菜真空渗入遗传转化法初报[J]. 西北农业大学学报, 1998(4):1-4.
	Zhang GH, Gong ZH, Xue WX, et al. Vacuum infiltration genetic transformation method in Chinese cabbage and rape[J]. J Northwest Agric Univ, 1998(4):1-4.
[11]	Liu F, Cao MQ, Yao L, et al. In planta transformation of pakchoi(Brassica campestris L. ssp. chinensis)by infiltration of adult plants with Agrobacterium[J]. Acta Hortic, 1998(467):187-192.
[12]	严继勇. BcpLH反义基因在大白菜中的转化及其功能的研究[D]. 杭州: 浙江大学, 2004.
	Yan JY. Studies on the function of BcpLH and transformation of its antisense gene into Chinese cabbage-pe-tsai(Brassica campestris L.)[D]. Hangzhou: Zhejiang University, 2004.
[13]	付绍红. floral-dip法转化甘蓝型油菜有关影响因素研究[D]. 雅安: 四川农业大学, 2005.
	Fu SH. A study on the related factors influencing transformation of Brassica napus by floral-dip[D]. Ya'an: Sichuan Agricultural University, 2005.
[14]	Xu HJ, Wang XF, Zhao H, et al. An intensive understanding of vacuum infiltration transformation of pakchoi(Brassica rapa ssp. chinensis)[J]. Plant Cell Rep, 2008, 27(8):1369-1376. doi: 10.1007/s00299-008-0564-3 URL
[15]	He YK, Bai JJ, Wu FJ, et al. In planta transformation of Brassica rapa and B. napus via vernalization-infiltration methods[J]. Protocol exchange, 2013. DOI:10.1038/protex.2013.067. doi: 10.1038/protex.2013.067
[16]	Chen GH, Zeng FL, Wang J, et al. Transgenic Wucai(Brassica campestris L.)produced via Agrobacterium-mediated anther transformation in planta[J]. Plant Cell Rep, 2019, 38(5):577-586. doi: 10.1007/s00299-019-02387-0 URL
[17]	Hu D, Bent AF, Hou XL, et al. Agrobacterium-mediated vacuum infiltration and floral dip transformation of rapid-cycling Brassica rapa[J]. BMC Plant Biol, 2019, 19(1):246. doi: 10.1186/s12870-019-1843-6 URL
[18]	Xiong XP, Liu WM, Jiang JX, et al. Efficient genome editing of Brassica campestris based on the CRISPR/Cas9 system[J]. Mol Genet Genomics, 2019, 294(5):1251-1261. doi: 10.1007/s00438-019-01564-w URL
[19]	Chen YY, Wang ZP, Ni HW, et al. CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis[J]. Sci China Life Sci, 2017, 60(5):520-523. doi: 10.1007/s11427-017-9021-5 URL
[20]	Tian SW, Jiang LJ, Gao Q, et al. Efficient CRISPR/Cas9-based gene knockout in watermelon[J]. Plant Cell Rep, 2017, 36(3):399-406. doi: 10.1007/s00299-016-2089-5 pmid: 27995308