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

doi:10.7668/hbnxb.20193230

Abstract

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

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.

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Figures/Tables 15

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

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/

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.

Fig.2 Sequences of SpSBP1 cDNA and predicted ORF

Fig.3 Conservative domain of SpSBP1

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

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

Fig.5 Phylogenetic tree of SpSBP1 homologous protein

Fig.6 Hydrophilic analysis of SpSBP1 protein

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

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.

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.

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

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.

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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