Cloning and Functional Prediction of Castor PIP5K2 Gene

doi:10.7668/hbnxb.20194529

Abstract

Abstract:

In plants,the PIP5K2 gene of the phosphatidylinositol 4,5-bisphosphate PIP5K gene family plays a key role in regulating plant growth.To investigate the function of the PIP5K2 gene in castor gene cloning,bioinformatics analysis,and expression analysis of the castor PIP5K2 gene were conducted.The results showed that a gene fragment of 2 136 bp in length was obtained through PCR using castor cDNA as the template.Bioinformatics analysis of the protein sequence encoded by this gene determined that the PIP5K2 gene encoded a protein consisting of 672 amino acids,with a pI value of 6.74 and a molecular weight of 76.47 ku.The protein's average hydrophilicity was -0.636,classifying it as a hydrophilic protein.Its secondary structure included α-helices,β-turns,extended strands,and random coils.Based on the prediction results,the tertiary structure of the PIP5K2 protein was consistent with its secondary structure,and shared a high degree of homology with the Jatropha curcas.The relative expression levels of the PIP5K2 gene were generally high in the five-leaf stage of the female,marker female and bisexual inflorescence types,and were generally low in the main stem panicle flowering stage.The highest relative expression level was observed in the five-leaf stage of the marker female inflorescence,while the lowest relative expression level was observed in the main stem panicle flowering stage of the bisexual inflorescence.The highest relative expression level was approximately 80 times higher than the lowest relative expression level.Among the three inflorescence types,the relative expression level of the PIP5K2 gene was similar in the four-leaf stage.However,compared to different growth stages within each inflorescence type,the expression level of the PIP5K2 gene was significantly higher in the four-leaf stage than in the flowering stage.Based on these results,it can be inferred that the PIP5K2 gene may regulate the mid-stage growth of castor plants and have some correlation with plant dwarfing.

Key words: Castor, PIP5K2 gene, Bioinformatics analysis, Plant regulation, Expression

YIN Mingda, LUO Rui, REN Wenjing, WANG Zhiyan, SU Zhimin, LI Ruxin, CHEN Zhen, LI Yuling, WANG Yan, HUANG Fenglan. Cloning and Functional Prediction of Castor PIP5K2 Gene[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(4): 72-79. doi: 10.7668/hbnxb.20194529.

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Figures/Tables 14

Tab.1 Biological information analysis softwares

软件 Softwares	网址 Websites	预测项目 Forecast project
NCBI	https://www ncbi nlm nih.gov/	序列下载
Protparam	https://web.expasy.org/protparam/1	蛋白质理化性质分析
TMHMM	http://www.cbs.dtu.dk/services/TMHMM	跨膜区分析
ExPASy	https://web.expasy.org/cgi-bin/protscale/	疏水性分析
SignalP 3.0	http://www.cbs.dtu.dk/services/SignalP/	信号肽预测
SMART	http://smart.embl-heidelberg.de/	蛋白质结构功能域分析
SOPMA	https://npsa-prabi.ibcp.fr/	蛋白质二级结构预测
SWISS Model	http://www.swissmodel.expasy.org/	蛋白质三级结构预测

Tab.2 Primer information of RT-qPCR

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
18S-F	AGGGGATAACCACCCCATGAATCCA
18S-R	TGCATGGTCTCCTGATACGGCCAAG
PK2-1F	TTAATTTTGTGTTTCGGC
PK2-1R	AGCAAGTGATAACCTCGC
PK2-2F	GACGCTTCTCCGTATCT
PK2-2R	TGCCTTTTCTCCACTCT

Fig.1 Castor tender inflorescence total RNA M.DL2000 Marker;1—3.Castor tender inflorescence total RNA.

Fig.2 Results of PIP5K2 gene PCR amplification electrophoresis M.DL2000 Marker;1,2.Amplification products of PCR.

Fig.3 Results of bacterial liquid PCR electrophoresis M.DL2000 Marker;1—6.Bacterial liquid PCR.

Fig.4 Electrophoresis results of vector plasmid digestion products M.DL5000 Marker;1.Control group;2.The results of enzyme digestion were verified.

Fig.5 Gene PIP5K2 sequencing results Query.Sequencing results;Sbjct.Original sequence.

Fig.6 Hydrophilicity prediction of PIP5K2 protein

Fig.7 Transmembrane region analysis of PIP5K2 protein

Fig.8 Secondary structure prediction of PIP5K2 protein

Fig.9 Tertiary structure prediction of PIP5K2 protein

Fig.10 Evolutionary tree analysis of proteins encoded by PIP5K2 gene in castor

Fig.11 Results of the Lm type female castor aLmAB5 inflorescence total RNA M.DL2000 Marker;1—12.Total RNA extracted from the inflorescences of the Lm type female castor aLmAB5,including four-leaf stage,five-leaf stage, main stem panicle flowering stage and secondary branch flowering stage from female,marker female and bisexual inflorescences types,totaling 12 samples.

Fig.12 Difference expression of PIP5Ks family genes in different types of plants 4D.Four-leaf stage of female;5D.Five-leaf stage of female;ZD.Main stem panicle flowering stage of female;ERD.Secondary branch flowering stage of female;4B.Four-leaf stage of iconic female;5B.Five-leaf stage of marker female;ZB.Main stem panicle flowering stage of marker female;ERB.Secondary branch flowering stage of marker female;4L.Four-leaf stage of bisexual;5L.Five-leaf stage of bisexual;ZL.Main stem panicle flowering stage of bisexual;ERL.Secondary branch flowering stage of bisexual.Different lowercase letters indicate significant differences(P<0.05).

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