Cloning of DoIPTs Genes in Yam and Its Expression Analysis During Bulbil Germination

doi:10.7668/hbnxb.20192488

Abstract

Abstract:

The aim of this research was to clone IPT(Isopentenyl transferase,IPT ) genes in yam,predict the structure and property of DoIPT proteins and identify the expression of DoIPTs during bulbil sprouting.DoIPTs were cloned by homologous cloning techniques.The structure and property of DoIPT proteins were predicted by bioinformatics analysis.Real-time PCR technique was applied to identify the expression of these genes.Three nucleic acid sequences of 1 398,1 044,1 349 bp were obtained from yam.The three sequences were named DoIPT1,DoIPT5a and DoIPT5b,and the registration numbers of these genes were MW353160,MW353016 and MW353017,respectively.The reading frame lengths were 1 137,861,1 011 bp respectively,and the encoded amino acid lengths were 378,286,336 aa,respectively.Bioinformatics analysis showed that DoIPTs were unstable,hydrophilic and external family proteins.There were two signal peptides in DoIPT5b,and no signal peptide was found in the other two proteins.The secondary structures in the three proteins were consistent with the prediction models of the tertiary structures of three genes.The encoded regions of DoIPTs had high similarity with the isopentenyl transferase protein of other plants.Coding areas of DoIPTs showed that the N-terminal regions of DoIPTs contained the P-loop NTPase structural domain GXXGXGKS(T),which was able to catalyse cytokinin synthesis.The results of Real-time PCR technique showed that the expression of DoIPT5a in sprouting bulbils was highest under the condition of water shortage at 22 ℃;and the expression of DoIPT1 and DoIPT5b genes was the highest in a humid environment;the expression of DOIPTs genes raised with paclobutrazol treatment.These results laid a foundation for further study on the function of DoIPTs in yam.

Key words: Yam, Gene cloning, DoIPT gene, Bulbil, Paclobutrazol(PP₃₃₃), Relative quantitative expression

TANG Wenfang, XU Shengsheng, DUAN Yanbi, LONG Wenhong, YANG Rongping, ZHANG Xuemei, MENG Jingui. Cloning of DoIPTs Genes in Yam and Its Expression Analysis During Bulbil Germination[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 27-34. doi: 10.7668/hbnxb.20192488.

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Figures/Tables 8

Tab.1 Primers used for PCR

引物名称 Primer name	用途 Purpose	序列(5'—3') Primer sequence	扩增大小/bp Amplification length
DoIpt1	中间扩增	F: ATTCACTGTTGGTTCCCTTCTG	1 120
		R: AGGCTTCGACCTGCTTGTCT
	5'RACE	①:CGATGGACGGAGGAGTCTTGT	279
		②:GATGTCAAGCCCTCTATAAACCTG
	3'RACE	①:AGACGGCTGTGGCGGAGAT	56
		②:GAAGATCAAGCGATTGGTGGAG
	全长验证	F: GGCAAAGAGAAGCAGAAC	1 315
		R:GACAGCGATAACCTGACA
	qRT-PCR	F:TGCCGCATCATCTCCTCG	221
		R:ATACCTGAACCGCTCTCGC
DoIpt5a	中间扩增	F:ACCTCGCCCTCCACTTCA	1 044
		R:CACCGCCTTCATCATACA
	全长验证	F:GTGTCGCTATAATTGTTCT	1 026
		R:AGGTTGATAGCCCAAGATA
	qRT-PCR	F:GTTGAGGAAGCAAGAGGAGTG	237
		R:ATCCCACCCTGACTCCACC
DoIpt5b	中间扩增	F: CCCTTTCCACTTGAATCCC	1 108
		R: AATGGCTCCACGCACTAAC
	5'RACE	①:GAGAAGTGCTTGGCTAAGTCG	182
		②:GAATCTCGTCTATGGCTTTCAT
	3'RACE	①:CGAATGTTACTTCCTTTGGGTC	202
		②:ACACGCTGGCTTTGCTTC
	全长验证	F: GTGAGGGTGTAAGAGTCAA	1 315
		R:GGAGTTTAGAGCCAAGTATG
	qRT-PCR	F:GAATCCCACCACCTTCTCCA	178
		R:CTCGTCGGTGACTTTGTTGG
RACE	5'Outer	CATGGCTACATGCTGACAGCCTA
	5'Inner	CGCGGATCCACAGCCTACTGATGATCAGTCGATG
	3'RT	ACGACTCACTATAGGGCN
CHC	qRT-PCR	F: GGTGAACTGTATGAGGCGG	156
		R: GCATCCACACAAGCAAAGCA

Fig.1 Gel electrophoresis map of RNA,DoIPTs full-length,and ORF encoding region of DoIPTs gene

Fig.2 Signal peptide of DoIPTs amino acids

Fig.3 Secondary structure and tertiary structure of DoIPTs proteins
A:Blue.α-helix;Red.Extended chain;Green.β-turn;Orange.Random curl.

Fig.4 Sequence alignment of DoIPTs amino acids
Similarity of 100%,100%—75%,75%—50%,and 0—33% was displayed with pink,blue,green and white,respectively;The binding site GXXGXGK(X standed for any amino acid)of ATP/GTP was displayed with red horizontal line.

Fig.5 Evolutionary tree of amino acids sequence of DoIPTs
Branches and leaves in phylogenetic tree were represented by protein name and registration number,and the first two letters of the protein name were abbreviated from the Latin name of the species.Ananas comosus.AcIPT5;Arabidopsis thaliana.AtIPT(1—9)9 proteins;Apostasia shenzhenica.AsIPT5;Brassica rapa.BrIPT3;Elaeis guineensis.EgIPT5;Helianthus annuus.HaIPT3;Hevea brasiliensis.HbIPT3;Manihot esculenta.MeIPT3;Morella rubra.MrIPT3;Phoenix dactylifera.PdIPT5;Theobroma cacao.TcIPT3;Triticum dicoccoides.TdIPT5.The proteins obtained in this study were marked with four diamonds.

Fig.6 Relative expression level of DoIPTs in bulbils at different periods
Fig.A shows the expression level of genes in the epidermis of bulbils when bulbils used their own water to regulate sprouting;Fig.B shows the gene expression level in the epidermis of bulbils after 60 days of bulbils harvest and application of water.Different lowercase letters indicate significant difference(Duncan's method,P<0.05).The same as Fig.7.

Fig.7 Relative expression level of DoIPTs under different concentrations of PP₃₃₃

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