Cloning and Expression Characteristics Analysis of RcNAC100-like Gene in Castor

doi:10.7668/hbnxb.20194389

Abstract

Abstract:

The NAC(NAM,ATAF1/2,CUC2)gene is an important regulatory factor in the stress signal transduction network.Cloning the NAC gene in castor,studying its molecular characteristics and expression characteristics,aiming to provide data support for the potential function of the castor NAC gene.The RcNAC100-like gene of Tongbi No.5 was cloned by RT-PCR technology,and its molecular characteristics were analyzed,including bioinformatics,subcellular localization,expression patterns,and transcription activation domain analysis.The results showed that the full length of RcNAC100-like gene cDNA was 1 244 bp,including a 1 086 bp coding sequence(CDS),encoding 361 amino acids.The protein had more irregular coil and α-helix structures,and was a hydrophilic,non-secretory protein.Phylogenetic analysis showed that the RcNAC100-like protein was most closely related to NAC proteins in Manihot esculenta and Hevea brasiliensis,with highly similar motif composition and positioning.The subcellular localization of RcNAC100-like protein was consistent with the predicted results,located in the nucleus.The predicted cis-acting elements in the RcNAC100-like promoter region indicate the presence of multiple environmental response and growth-related elements.Expression pattern analysis showed that the RcNAC100-like gene had tissue-specific expression,with the highest relative expression level in the roots.Additionally,the gene could respond rapidly to adverse environments(drought,salt,cold,and ABA stress)and actively express,indicating that the RcNAC100-like gene might be a key gene in the castor's response to stress.Transcription activation assay results showed that the RcNAC100-like transcription factor has transcriptional activation activity in yeast.In summary,the RcNAC100-like gene may play an important role in the castor's resistance to adversity.

Key words: Castor, RcNAC100-like gene, Bioinformatics, Clone, Expression

LI Yanxiao, WANG Lina, ZHU Guishuang, LIU Peng, XIANG Dianjun. Cloning and Expression Characteristics Analysis of RcNAC100-like Gene in Castor[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 67-76. doi: 10.7668/hbnxb.20194389.

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Figures/Tables 12

Tab.1 Primer sequences and uses

引物名称 Primer names	描述 Description	引物序列(5'—3') Primer sequence(5'—3')
RcNAC100-like	基因克隆	F:CAAGAGAACAGTAGGGTTTTAGC
		R:CAGCGAGCCAAACCATTTTTC
q-RT-RcNAC100-like	RcNAC100-like表达水平	F:CTCTATCCACAACCTCCCCA
		R:CCGAAAGAACCTAACCTCAC
q-RT-Actin	Actin表达水平	F:TCCCTCAGTACGTTCCAGCA
		R:CACCTCCATACTCCTCCCT
P-RcNAC100-like	启动子克隆	F:TATATAATGCTTATTTTTAATATAG
		R:TTCTTTAAAGAGAAGAAAACGAGAG
35S∷RcNAC100-like-GFP	亚细胞定位	F:AGAACACGGGGGACGGATCCATGGAAAACATTTCTGGTCTTG(BamH Ⅰ)
		R:ACCATGGTGTCGACGTCGACGTAGTTCCAAAGACAATCAAGGTCC(Sal Ⅰ)
RcNAC100-like:1~423	转录激活活性检测	F:CATATGATGGAAAACATTTCTGGTCTTG(Nde Ⅰ)
		R:GGATCCTAATCTGTATTCATGCATGACCC(BamH Ⅰ)
RcNAC100-like:424~1 086	转录激活活性检测	F:CATATGGAGGGTAAATACTCTATCCAC(Nde Ⅰ)
		R:GGATCCTCAGTAGTTCCAAAGACAATC(BamH Ⅰ)

Fig.1 Cloning of RcNAC100-like gene 1.RcNAC100-like gene;M.DL2000 Marker.

Fig.2 The deduced amino acid sequence of RcNAC100-like gene

Fig.3 Location,collinearity and structural domain analysis of RcNAC100-like gene on chromosome A.Location and collinearity of the RcNAC100-like gene;B.Structural domain prediction of the RcNAC100-like protein.

Fig.4 Prediction of phosphorylation sites for RcNAC100-like protein

Fig.5 Structural analysis of RcNAC100-like protein A.Transmembrane domain;B.Signal peptide structure;C.Secondary structure;D.Tertiary structure(left)and pull-type conformation scoring diagram(right).

Fig.6 Evolution and conserved motif analysis of RcNAC100-like proteins A.Evolutionary tree analysis of NAC proteins;B.Conserved motif analysis;C.Structural domain analysis.

Fig.7 Analysis of cis-acting elements in RcNAC100-like promoter A.RcNAC100-like promoter clone:1.RcNAC100-like promoter;M.DL2000 Marker.B.RcNAC100-like cis-acting element prediction.

Fig.8 Subcellular localization of RcNAC100-like protein

Fig.9 Tissue expression profile of the RcNAC100-like gene

Fig.10 The abiotic stress expression pattern of RcNAC100-like gene Different lowercase letters indicate significant differences among treatments(P<0.05).

Fig.11 Analysis of RcNAC100-like transcriptional activity A.pGBKT7,pGBKT7-FL,pGBKT7-C,and pGBKT7-N represent the empty vector, RcNAC100-like full-length(1—1 086 bp),N-terminal(1—423 bp),and C-terminal(424—1 086 bp)subcloning schematic,respectively;B.From left to right are the positions of each vector on the plate,SD/-Trp medium,and SD/-Ade/-His/-Trp+ X-α-gal medium.

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