杜梨PbGID1家族基因克隆、CRISPR/Cas9载体构建及遗传转化

doi:10.7668/hbnxb.20193553

摘要/Abstract

摘要：

为明确杜梨赤霉素受体(GID1)的生物学功能,以杜梨为试材,通过同源克隆方法得到PbGID1s基因,利用生物信息学分析软件构建基因结构并设计靶位点;利用酶切-连接方法将带有靶点的sgRNA表达盒构建至CRISPR/Cas9表达载体上;通过农杆菌介导方法,将CRISPR/Cas9表达载体转化至杜梨子叶中。结果表明,在杜梨基因组中克隆得到4个PbGID1s,分别命名为PbGID1b-1、PbGID1b-2、PbGID1c-1、PbGID1c-2,基因结构分析显示该家族基因均由2个外显子和1个内含子组成;氨基酸序列比对发现,PbGID1s均具有GID1家族所特有的HGG和GXSXG保守结构域;将含有5个靶位点的sgRNA表达盒成功构建至pYLCRISPR/Cas9P_35S-N植物表达载体上,可同时对该家族4个基因进行编辑;杜梨遗传转化试验结果表明,共计浸染595粒杜梨子叶,获得抗性芽176个,阳性植株33株,转化效率达到5.55%。成功构建了可以同时靶向杜梨PbGID1s家族基因的CRISPR/Cas9载体,通过农杆菌介导,成功将该载体转化至杜梨子叶,并获得阳性植株。

关键词: 杜梨, GA受体, GID1, CRISPR/Cas9, 遗传转化

Abstract:

In order to clarify the biological function of gibberellin receptor GID1 in Pyrus betulifolia,and provide a good foundation for future development of P.betulifolia dwarf rootstocks using CRISPR/Cas9 gene editing technology.Pyrus betulifolia was used as the test material,and the PbGID1s genes were obtained by homologous cloning method.Bioinformatics analysis software was used to construct the gene structure and design the target sites;construction of sgRNA expression cassettes with targets into CRISPR/Cas9 expression vectors,through the mediation of Agrobacterium,the CRISPR/Cas9 expression vector was transferred into the cotyledons of P.betulifolia.Results showed that four PbGID1s were successfully cloned from P.betulifolia plants and named as PbGID1b-1,PbGID1b-2,PbGID1c-1 and PbGID1c-2. They all consisted of two exons and one intron found by gene structure analysis.Amino acid sequence comparison showed that all PbGID1s had the HGG and GXSXG conserved domains.Five gRNAs that could potentially edit all 4 PbGID1s simultaneously were successfully constructed into a single CRISPR/Cas9 vector,pYLCRISPR/Cas9P_35S-N.The results of the genetic transformation test of P.betulifolia showed that a total of 595 cotyledons of P.betulifolia were infiltrated,176 resistant buds and 33 positive plantlets were obtained,and the transformation efficiency reached 5.55%.A CRISPR/Cas9 vector was successfully constructed that could simultaneously target the PbGID1s family genes of P.betulifolia.Through the mediation of Agrobacterium,the vector was successfully transformed into P.betulifolia cotyledons,and positive plants were obtained.

Key words: Pyrus betulifolia, GA receptor, GID1, CRISPR/Cas9, Genetic transformation

宋平丽, 李刚, 许建锋, 马青翠, 亓宝秀, 张玉星. 杜梨PbGID1家族基因克隆、CRISPR/Cas9载体构建及遗传转化[J]. 华北农学报, 2022, 37(6): 15-22. doi: 10.7668/hbnxb.20193553.

SONG Pingli, LI Gang, XU Jianfeng, MA Qingcui, QI Baoxiu, ZHANG Yuxing. Cloning of PbGID1 Family Genes from Pyrus betulifolia Bunge,CRISPR/Cas9 Gene Editing Vector Construction and Genetic Transformation[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 15-22. doi: 10.7668/hbnxb.20193553.

http://www.hbnxb.net/CN/Y2022/V37/I6/15

图/表 10

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引物名称 Primer name	序列(5'—3') Sequence(5'—3')
PbGID1bF	ATGGCTGGTAGTAATGAAG
PbGID1b-1R	TTAACAGTCAGGATTCACGAAGT
PbGID1b-2R	TTAACAGTCAGGATTCACGAAGC
PbGID1c-1F	ATGTGTTGGGTTTTCTTCAAG
PbGID1c-1R	TCAATAGTTGGAGCACACG
PbGID1c-2F	ATGTCTGGGGGCAATGAAG
PbGID1c-2R	TCAATTGTCGGAGAGCACG

引物名称 Primer name	序列(5'—3') Sequence(5'—3')
PbGID1bF	ATGGCTGGTAGTAATGAAG
PbGID1b-1R	TTAACAGTCAGGATTCACGAAGT
PbGID1b-2R	TTAACAGTCAGGATTCACGAAGC
PbGID1c-1F	ATGTGTTGGGTTTTCTTCAAG
PbGID1c-1R	TCAATAGTTGGAGCACACG
PbGID1c-2F	ATGTCTGGGGGCAATGAAG
PbGID1c-2R	TCAATTGTCGGAGAGCACG

引物 Primer	序列(5'—3') Sequence(5'—3')
U-F	CTCCGTTTTACCTGTGGAATCG
gR-R	CGGAGGAAAATTCCATCCAC
gRT1	GCACACTCCTCTTCAAACAGGTTTTAGAGCTAGAAAT
AtU3dT1	CTGTTTGAAGAGGAGTGTGCTGACCAATGGTGCTTTG
gRT2	GTACACTCCTCTTCAAACAGGTTTTAGAGCTAGAAAT
AtU3bT2	CTGTTTGAAGAGGAGTGTACTGACCAATGTTGCTCC
gRT3	ATACTGCTCAACCCTATGTTGTTTTAGAGCTAGAAAT
AtU6-1T3	AACATAGGGTTGAGCAGTATCAATCACTACTTCGTCT
gRT4	CCTTGTCAACACTTGCAAGGGTTTTAGAGCTAGAAAT
AtU6-29T4	CCTTGCAAGTGTTGACAAGGCAATCTCTTAGTCGACT
gRT5	GCATTTCTACTGCCTCATGGGTTTTAGAGCTAGAAAT
AtU3bT5	CCATGAGGCAGTAGAAATGCTGACCAATGTTGCTCC
gRT6	TGTAACCCATTTGGTCCCCGGTTTTAGAGCTAGAAAT
AtU6-1T6	CGGGGACCAAATGGGTTACACAATCACTACTTCGTCT
gRT7	CCACGTCGCGGTCAAGGCGGGTTTTAGAGCTAGAAAT
AtU6-29T7	CCGCCTTGACCGCGACGTGGCAATCTCTTAGTCGACT
gRT8	GCAGCCCTTGAGCACCACCGGTTTTAGAGCTAGAAAT
AtU3dT8	CGGTGGTGCTCAAGGGCTGCTGACCAATGGTGCTTTG
Pps-GGL	TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGC
	AAAGG
Pgs-GG2	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
Pps-GG2	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
Pgs-GG3	AGCGTGGGTCTCGTCTTCACTCCATCCACTCCAAGCTC
Pps-GG3	TTCAGAGGTCTCTAAGACTTTGGAATCGGCAGCAAAGG
Pgs-GG4	AGCGTGGGTCTCGAGTCCTTTCCATCCACTCCAAGCTC
Pps-GG4	TTCAGAGGTCTCTGACTACATGGAATCGGCAGCAAAGG
Pgs-GG5	AGCGTGGGTCTCGGTCCACATCCATCCACTCCAAGCTC
Pps-GG5	TTCAGAGGTCTCTGGACTAGTGGAATCGGCAGCAAAGG
Pgs-GG6	AGCGTGGGTCTCGCAGATAGTCCATCCACTCCAAGCTC
Pps-GG6	TTCAGAGGTCTCTTCTGCAATGGAATCGGCAGCAAAGG
Pgs-GG7	AGCGTGGGTCTCGACCTCAATCCATCCACTCCAAGCTC
Pps-GG7	TTCAGAGGTCTCTAGGTTTCTGGAATCGGCAGCAAAGG
Pgs-GG8	AGCGTGGGTCTCGAGCGTTCTCCATCCACTCCAAGCTC
Pps-GG8	TTCAGAGGTCTCTCGCTGATTGGAATCGGCAGCAAAGG
Pgs-GGL	AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAA
	GCTC
SP-L2	GTCGTGCTCCACATGTTGACCG
SP-R-C	CCGACATAGATGCAATAACTT

引物 Primer	序列(5'—3') Sequence(5'—3')
U-F	CTCCGTTTTACCTGTGGAATCG
gR-R	CGGAGGAAAATTCCATCCAC
gRT1	GCACACTCCTCTTCAAACAGGTTTTAGAGCTAGAAAT
AtU3dT1	CTGTTTGAAGAGGAGTGTGCTGACCAATGGTGCTTTG
gRT2	GTACACTCCTCTTCAAACAGGTTTTAGAGCTAGAAAT
AtU3bT2	CTGTTTGAAGAGGAGTGTACTGACCAATGTTGCTCC
gRT3	ATACTGCTCAACCCTATGTTGTTTTAGAGCTAGAAAT
AtU6-1T3	AACATAGGGTTGAGCAGTATCAATCACTACTTCGTCT
gRT4	CCTTGTCAACACTTGCAAGGGTTTTAGAGCTAGAAAT
AtU6-29T4	CCTTGCAAGTGTTGACAAGGCAATCTCTTAGTCGACT
gRT5	GCATTTCTACTGCCTCATGGGTTTTAGAGCTAGAAAT
AtU3bT5	CCATGAGGCAGTAGAAATGCTGACCAATGTTGCTCC
gRT6	TGTAACCCATTTGGTCCCCGGTTTTAGAGCTAGAAAT
AtU6-1T6	CGGGGACCAAATGGGTTACACAATCACTACTTCGTCT
gRT7	CCACGTCGCGGTCAAGGCGGGTTTTAGAGCTAGAAAT
AtU6-29T7	CCGCCTTGACCGCGACGTGGCAATCTCTTAGTCGACT
gRT8	GCAGCCCTTGAGCACCACCGGTTTTAGAGCTAGAAAT
AtU3dT8	CGGTGGTGCTCAAGGGCTGCTGACCAATGGTGCTTTG
Pps-GGL	TTCAGAGGTCTCTCTCGACTAGTATGGAATCGGCAGC
	AAAGG
Pgs-GG2	AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC
Pps-GG2	TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG
Pgs-GG3	AGCGTGGGTCTCGTCTTCACTCCATCCACTCCAAGCTC
Pps-GG3	TTCAGAGGTCTCTAAGACTTTGGAATCGGCAGCAAAGG
Pgs-GG4	AGCGTGGGTCTCGAGTCCTTTCCATCCACTCCAAGCTC
Pps-GG4	TTCAGAGGTCTCTGACTACATGGAATCGGCAGCAAAGG
Pgs-GG5	AGCGTGGGTCTCGGTCCACATCCATCCACTCCAAGCTC
Pps-GG5	TTCAGAGGTCTCTGGACTAGTGGAATCGGCAGCAAAGG
Pgs-GG6	AGCGTGGGTCTCGCAGATAGTCCATCCACTCCAAGCTC
Pps-GG6	TTCAGAGGTCTCTTCTGCAATGGAATCGGCAGCAAAGG
Pgs-GG7	AGCGTGGGTCTCGACCTCAATCCATCCACTCCAAGCTC
Pps-GG7	TTCAGAGGTCTCTAGGTTTCTGGAATCGGCAGCAAAGG
Pgs-GG8	AGCGTGGGTCTCGAGCGTTCTCCATCCACTCCAAGCTC
Pps-GG8	TTCAGAGGTCTCTCGCTGATTGGAATCGGCAGCAAAGG
Pgs-GGL	AGCGTGGGTCTCGACCGACGCGTATCCATCCACTCCAA
	GCTC
SP-L2	GTCGTGCTCCACATGTTGACCG
SP-R-C	CCGACATAGATGCAATAACTT

试剂 Reagents	加入量 Volume
10×CutSmart Buffer	3 μL
10 mmol/L ATP	3 μL
pYLCRISPR/Cas9质粒	160 ng
pYLCRISPR/Cas9 plasmid
目标靶点sgRNA表达盒	每个表达盒30 ng
Target sgRNA cassettes
Bsa Ⅰ-HF	1 μL
T₄ DNA ligase	0.2 μL
H₂O	补足至30 μL

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