马铃薯野生种<i>SpSBP1</i>基因的克隆及CRISPR/Cas9载体构建

doi:10.7668/hbnxb.20193230

摘要/Abstract

摘要：

为了探究SBP1基因在植物自交不亲和方面的重要作用,以二倍体马铃薯野生种为材料,利用RT-PCR技术克隆获得马铃薯野生种 SpSBP1(登录号:MZ803088)的cDNA全长序列,并对其进行生物信息学分析及 CRISPR/Cas9载体构建。结果显示,SpSBP1基因的cDNA全长为1 176 bp,包含92 bp的5'非编码区和163 bp的3'非编码区,其最大开放阅读框为921 bp,编码306个氨基酸。蛋白结构域分析显示,SpSBP1蛋白包括Smc超家族结构域以及RING finger和Zinc finger结构域。蛋白同源序列比对及系统进化树分析显示,马铃薯野生种SpSBP1与马铃薯野生种ScSBP1的同源性最高,其次为花烟草。生物信息学分析显示,SpSBP1分子质量为34.731 44 ku,理论等电点pI为5.10,推测得出该蛋白为酸性不稳定亲水性蛋白。二级结构预测该蛋白主要由α螺旋和无规则卷曲所构成。同时该蛋白属于非分泌蛋白且不存在跨膜结构。从二倍体马铃薯野生种中克隆出了SpBP1基因,同时成功构建了CRISPR/Cas9载体,并进行了遗传转化试验。

关键词: 马铃薯野生种, 自交不亲和, SpSBP1, 克隆, 生物信息学分析, CRISPR/Cas9载体构建

Abstract:

In order to explore the important role of SBP1(S-RNase-binding protein 1)gene in plant self-incompatibility,a diploid wild potato was used as the material to obtain the full-length cDNA sequence of potato SpSBP1 (GenBank: MZ803088)by using RT-PCR cloning technique,and performed bioinformatics analysis and CRISPR/Cas9 vector construction for SpSBP1 gene.The results showed that the full length cDNA of SpSBP1 gene was 1 176 bp,containing 92 bp 5' non-coding region and 163 bp 3' non-coding region.The maximum open reading frame(ORF)of SpSBP1 gene was 921 bp,which encoding 306 amino acids.Protein domain analysis showed that SpSPB1 protein included Smc superfamily domain,RING finger domain and Zinc finger domain.The homologous sequence alignment and phylogenetic tree analysis showed that SpSBP1 had the highest homology with S.chacoense,followed by Nicotiana alata.Bioinformatics analysis showed that the molecular weight of SpSBP1 was 34.731 44 ku,with a theoretical isoelectric point of 5.10,and it was speculated that SpSBP1 was an acid unstable hydrophilic protein. The secondary structure predicted that the protein was mainly composed of α helix and random curling. And the protein was a non-secretory protein and had no transmembrane structure. SpSBP1 gene was cloned from wild diploid potato,and CRISPR/Cas9 vector was successfully constructed,and also the genetic transformation was carried out.

Key words: Solanum pinnatisectum, Self-incompatibility, SpSBP1, Cloning, Bioinformatics analysis, CRISPR/Cas9 vector construction

陈娜, 邵勤, 李晓鹏, 高阳, 卢其能. 马铃薯野生种SpSBP1基因的克隆及CRISPR/Cas9载体构建[J]. 华北农学报, 2022, 37(6): 23-33. doi: 10.7668/hbnxb.20193230.

CHEN Na, SHAO Qin, LI Xiaopeng, GAO Yang, LU Qineng. Cloning and CRISPR/Cas9 Vector Construction of SpSBP1 Gene in Potato[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 23-33. doi: 10.7668/hbnxb.20193230.

http://www.hbnxb.net/CN/Y2022/V37/I6/23

图/表 15

表1 引物序列

Tab.1 Primer sequences

引物 Primer	引物序列(5'—3') Sequence (5'—3')	用途 Application
SpSBP1-F	CGCTTCCTCATCACCATC	基因克隆
SpSBP1-R	CCATCCAAGGAAGAACCC
pMD19-F	CGCCAGGGTTTTCCCAGTCACGAC	通用引物
pMD19-R	CGCCAGGGTTTTCCCAGTCACGAC
SpSBP1-CRISPR/Cas9-F	ATATATGGTCTCGTTTGGTTGGAGCCAAAGAAGAA	CRISPR/Cas9载体构建
	GGTTTTAGAGCTAGAAATAG
SpSBP1-CRISPR/Cas9-R	ATTATTGGTCTCGAAACATCGCCGCTGCAACAGATC
	CCAAACTACACTGTTAGATTC

表2 生物信息学分析网站

Tab.2 Bioinformatics analysis web site

工具Tools	网站Web site
NCBI-ORF	https://www.ncbi.nlm.nih.gov/orffinder/
NCBI-PROGRAM	https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
ClustalW2	https://www.ebi.ac.uk/Tools/msa/clustalw2/
ProtParam	https://web.expasy.org/protparam/
SOPMA	https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html
SignalP 5.0	http://www.cbs.dtu.dk/services/SignalP/
TMHMM Server v.2.0	http://www.cbs.dtu.dk/services/TMHMM/

图1 马铃薯野生种 SpSBP1基因 A: M.Marker DL2000,1.SpSBP1基因;B: M.Marker DL2000,1—3,6—8.阳性克隆,4—5.阴性克隆。

Fig.1 S.pinnatisectum SpSBP1 gene A: M.Marker DL2000,1.SpSBP1 gene;B: M.DL2000,1—3,6—8.Positive clone,4—5.Negative clone.

图2 SpSBP1 cDNA及预测的开放阅读框

Fig.2 Sequences of SpSBP1 cDNA and predicted ORF

图3 SpSBP1的保守区

Fig.3 Conservative domain of SpSBP1

表3 试验中各物种的SBP1基因

Tab.3 SBP1 genes of species in the experiment

编号 No.	物种 Species	SBP1基因 SBP1 gene
1	马铃薯野生种	ScSBP1
2	紫矮牵牛	PiSBP1
3	矮牵牛	PhSBP1
4	花烟草	NaSBP1
5	毛果杨	PtSBP1
6	苹果	MdSBP1
7	甜樱桃	PaSBP1
8	长蒴黄麻	CoSBP1
9	苜蓿	MtSBP1
10	拟南芥	AtSBP1
11	二倍体马铃薯野生种	SpSBP1

图4 马铃薯野生种SpSBP1编码的蛋白与其他物种同源蛋白多重序列比对

Fig.4 Multiple alignment of homologous protein for the SpSBP1 in S.pinnatisectum and other species

图5 SpSBP1同源蛋白的系统进化树

Fig.5 Phylogenetic tree of SpSBP1 homologous protein

图6 SpSBP1蛋白亲疏水性分析

Fig.6 Hydrophilic analysis of SpSBP1 protein

图7 SpSBP1编码的蛋白的二级结构预测

Fig.7 Secondary structure prediction of protein encoded by SpSBP1 gene

图8 SpSBP1基因编码的蛋白的信号肽和跨膜结构分析 A.SpSBP1基因编码的蛋白的信号肽预测;B.SpSBP1基因编码的蛋白的跨膜结构预测。

Fig.8 Signal peptide and transmembrane structure analysis of protein encoded by SpSBP1 gene A.Signal peptide prediction of protein encoded by SpSBP1 gene;B.Transmembrane structure prediction of protein encoded by SpSBP1 gene.

图9 马铃薯野生种SpSBP1基因的CRISPR/Cas9载体构建 A: M.Marker DL2000,1—2.SpSBP1基因;B: M.Marker DL2000,1—4,6—7.阳性克隆,5.阴性克隆。

Fig.9 CRISPR/Cas9 vector construction of S.pinnatisectum SpSBP1 gene A: M.Marker DL2000,1—2.SpSBP1 gene;B: M.Marker DL2000,1—4,6—7.Positive clones,5.Negative clones.

图10 马铃薯SpSBP1-CRISPR/Cas9载体示意图

Fig.10 Structural diagram of S.pinnatisectum SpSBP1-CRISPR/Cas9 vector

图11 马铃薯遗传转化过程 A.马铃薯幼苗;B.预培养;C.共培养;D.抑菌培养;E、F.筛选培养;G、H.生根培养;I.移栽。

Fig.11 Process of potato genetic transformation A.Potato plantlet in vitro;B.Pre-culture;C.Co-culture;D.Antibacterial culture;E,F.Screening culture;G,H.Rooting culture;I.Transplanting.

图12 部分马铃薯再生植株的PCR检测 M.Marker DL2000;CK+.阳性质粒对照;CK-.未转化植株对照;3,9,12,14—16,22—23,27—31.阳性植株;1—2,4—8,10—11,13,17—21,24—26,32.阴性植株。

Fig.12 PCR analysis of some potato regenerated plants M.Marker DL2000;CK+.Positive plasmid control;CK-.Untransformed plant control;3,9,12,14—16,22—23,27—31.Positive plants;1—2,4—8,10—11,13,17—21,24—26,32.Negative plants.

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[41]	韩政宏, 段宇轩, 徐善斌, 王敬国, 刘化龙, 杨洛淼, 贾琰, 辛威, 郑洪亮, 邹德堂. 利用CRISPR/Cas9技术敲除GS3和GS9基因改良水稻粒型性状[J]. 华北农学报, 2022, 37(2): 9-17. doi: 10.7668/hbnxb.20192762. doi: 10.7668/hbnxb.20192762
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马铃薯野生种SpSBP1基因的克隆及CRISPR/Cas9载体构建

Cloning and CRISPR/Cas9 Vector Construction of SpSBP1 Gene in Potato

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