Cloning and Expression Analysis of Cassava MeNAC29 and MeNAC30 Genes

doi:10.3969/j.issn.1000-2561.2019.04.012

Abstract

Abstract:

The NAC (NAM/ATAF/CUC) transcription factor gene family was widely distributed in terrestrial plants and plays an important role in the regulation of growth and stress responses. The preliminary research about the role of this gene family on the cassava against bacterial blight was carried out in our lab. Two genes, MeNAC29 and MeNAC30, were cloned from cassava cDNA by RT-PCR, the different expression changes between resistant and susceptible cassava germplasms infected by Xanthomonas axonopodis pv. manihotis (Xam) were also quantified by qRT-PCR. The results showed both MeNAC29 and MeNAC30 contain three exons, two introns, and conserved NAM domain. The length of MeNAC29 gene’s ORF was 888 nt, which encode 295 amino acids, while the MeNAC30 gene’s ORF was 870 nt and encode 289 amino acids. Compared with susceptible cassava germplasms, both two genes’ expression was significantly increased in the resistant cassava germplasms - Xanthomonas axonopodis pv. manihotis interaction, which showed they are involved in resistance response of cassava to bacterial blight.

Key words: cassava, MeNAC29 gene, MeNAC30 gene, expression analysis

LIN Zhaowei,LI Chaoping,SHI Tao,WANG Guofen,LI Boxun,CAI Jimiao,HUANG Guixiu. Cloning and Expression Analysis of Cassava MeNAC29 and MeNAC30 Genes[J]. Chinese Journal of Tropical Crops, 2019, 40(4): 701-707.

Figures/Tables 9

Tab. 1 Primer sequences and the application

引物名称Primer name	引物序列Primer sequence (5°-3')	用途Application
MeNAC29-F MeNAC29-R	AGGGATGCTATGGTCGTCA CCAGAGCGAGACTACAATAAGC	MeNAC29克隆
MeNAC30-F MeNAC30-R	CACAGCATTTCACCACAAAG AGGCACGAGACAACTACAAA	MeNAC30克隆
MeNAC29-QF MeNAC29-QR	TTCACTCTGGACCTCCTTCTC CATACGGACGCATCTGATTC	MeNAC29 qRT-PCR
MeNAC30-QF MeNAC30-QR	TGAGCCCAAGTGGAAAGAGT GCGGCGACATTTGATTATTC	MeNAC30 qRT-PCR
18S-QF 18S-QR	GCTCGAAGACGATCAGATACC TTCAGCCTTGCGACCATAC	qRT-PCR内参基因

Fig. 1 The sketch diagram of the MeNAC29 gene Black bars indicate exons; solid lines indicate sequentially the 5'-UTR, introns and the 3'-UTR.

Fig. 2 The sketch diagram of the MeNAC30 gene Black bars indicate exons; solid lines indicate sequentially the 5'-UTR, introns and the 3'-UTR.

Tab. 2 Amino acid composition of MeNAC29 and MeNAC30

蛋白质 Protein	氨基酸含量 Amino acid content /%
蛋白质 Protein	Ala	Arg	Asn	Asp	Cys	Gln	Glu	Gly	His	Ile	Leu
MeNAC29	5.4	4.7	5.1	8.1	1.4	4.7	5.8	6.1	2.7	5.4	8.1
MeNAC30	8.3	4.2	4.8	6.9	1.7	3.1	5.2	5.5	2.1	4.5	5.9
蛋白质 Protein	氨基酸含量 Amino acid content /%
蛋白质 Protein	Lys	Met	Phe	Pro	Ser	Thr	Trp	Tyr	Val	Pyl	Sec
MeNAC29	8.5	2.0	4.7	7.8	6.1	3.1	2.0	4.7	3.4	0.0	0.0
MeNAC30	8.3	3.1	4.2	9.0	7.3	4.2	2.4	5.2	4.2	0.0	0.0

Fig. 3 The putative secondary structure of MeNAC29 and MeNAC30 A: MeNAC29, B: MeNAC30; Blue: Alpha helix, Green: Beta turn, Purple: Random coil, Red: Extended strand.

Fig. 4 The predicted 3-dimensional structure of MeNAC29

Fig. 5 The predicted 3-dimensional structure of MeNAC30

Fig. 6 The phylogenetic tree of MeNAC29 and MeNAC30 and their homologous proteins

Fig. 7 qRT-PCR analysis of the changes in the expression of MeNAC29 and MeNAC30 between cassava resistant and susceptible germplasms following Xam inoculation Different lowercase letters represent significant difference among different germplasm at same inoculation time (P<0.05).

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