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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (12): 1914-1922.doi: 10.3724/SP.J.1006.2020.04006

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Cloning of the key enzyme gene HcTPPJ in trehalose biosynthesis of kenaf and its expression in response to abiotic stress in kenaf

Hui LI1,2(), De-Fang LI2,*(), Yong DENG2, Gen PAN2, An-Guo CHEN2, Li-Ning ZHAO2, Hui-Juan TANG2   

  1. 1College of Life and Environment Science, Hunan University of Arts and Science, Changde 415000, Hunan, China
    2Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, Hunan, China
  • Received:2020-01-10 Accepted:2020-08-19 Online:2020-09-08 Published:2020-11-25
  • Contact: De-Fang LI E-mail:guangjunmuzi@126.com;18973612200@163.com
  • Supported by:
    China Agriculture Research System(CARS-19-E07);Agricultural Science and Technology Innovation Program at the Chinese Academy of Agricultural Science(ASTIP-IBFC03);Hunan Education Department Project(18C0737);Doctoral Research Start-up Project of Hunan University of Arts and Sciences(17BSQD13)

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Abstract:

Trehalose biosynthesis key enzyme gene TPP plays an important role in plant response to various abiotic stresses. In this study, in order to clarify the role of TPP gene in response to abiotic stress in kenaf, a specific primer was designed according to the sequence of CL541.contig2unigene, and the full-length cDNA sequence of TPP gene was obtained by PCR. Bioinformatics analysis showed that TPP had an open reading frame (ORF) length of 1128 bp, and encoded a protein containing 375 amino acids. The results of amino acid sequence consistency indicated that the agreement between the amino acid sequence of protein and that of TPPJ from other species was 71.18%, so the gene named as HcTPPJ. HcTPPJ was expressed in roots, stems and leaves. Under salt or drought stress, HcTPPJ was up-regulated significantly with the extension of stress treatment, indicating that the gene was involved in the response process of salt or drought stress in kenaf. Under the same conditions, HcNCED3 and HcAOC was significantly up-regulated, while the change of HcTPPJ expression was not obvious under ABA stress; HcTPPJ was significantly down-regulated, under the stress of MeJA for six hours. Therefore it was speculated that the HcTPPJ gene expression may be not regulated by the signal molecule of ABA, but negatively regulated by methyl jasmonate signal molecule. This study will lay a solid foundation for further elucidating the role of the gene in response to salt and drought stress in kenaf.

Key words: kenaf, salt stress, trehalose-6-phosphate phosphatase, ABA

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequences (5°-3°)		 引物用途
Primer usage
HcTPPJ-F ATGGTGAGTTTCTTTGAA 基因克隆
HcTPPJ-R TTACATTTTAGATTGCCCT Gene cloning
HcTPPJ-QF AACCTTTCTGCCTTGAGT 实时荧光定量PCR
HcTPPJ-QR AAATTGGCTGAGCTGTAC qRT-PCR
HcNCED3-QR AGGCGGTCGTCGGACTCGTT 实时荧光定量PCR
HcNCED3-QF GACTGCTTCTGCTTCCACCTCTG qRT-PCR
Actin-QF CAGGCAGTTCTTTCTTTGT 内参基因
Actin- QR ATCCTCCAATCCAGACACT Reference gene

Fig. 1

Agarose gel electrophoresis of cDNA full length of HcTPPJ M: DNA marker 2K Plus II; 1: PCR product."

Fig. 2

Amino acid sequence alignment of HcTPPJ and TPPJ proteins from other plants OsTPP1: Oryza sativa; AtTPPA, AtTPPB, AtTPPD: Arabidopsis thaliana; TPPJ: Theobroma cacao, Hibiscus syriacus, Durio zibethinus, Gossypium raimondii, Populus trichocarpa, Hibiscus cannabinus, Hevea brasiliensis, Punica granatum. The different colors indicate that conservatism of different amino acid residues. The blue means amino acids are complete conservation, the pink, cyan, yellow mean that the conservation of amino acids is no less than 75%, no less than 50%, no less than 33%, respectively, and white mean no more than 33%."

Fig. 3

Phylogenetic tree of HcTPPJ and TPPJ from other plants TPPJ: Theobroma cacao, Hibiscus syriacus, Durio zibethinus, Gossypium raimondii, Populus trichocarpa, Hibiscus cannabinus, Hevea brasiliensis, Punica granatum; AtTPPA, AtTPPB, AtTPPD: Arabidopsis thaliana; OsTPP1: Oryza sativa."

Fig. 4

Expression of HcTPPJ gene in different organs * indicates significantly different at the 0.05 probability level. Error bars represent the standard error of each treating group (n = 3)."

Fig. 5

Expression of HcTPPJ in different organs after three days under 150 mmol L-1 NaCl stress * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively. Error bars represent the standard error of each treating group (n = 3)."

Fig. 6

Expression of HcTPPJ in leaves under different NaCl concentration stress * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively. Error bars represent the standard error of each treating group (n = 3)."

Fig. 7

Expression of HcTPPJ in leaves under 150 mmol L-1 NaCl stress at different times ** indicate significantly different at the 0.01 probability level. Error bars represent the standard error of each treating group (n = 3)."

Fig. 8

Expression of HcTPPJ in different organs under drought stress 8 h (A) and expression of HcTPPJ in leaves under drought stress at different times (B) * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively. Error bars represent the standard error of each treating group (n = 3)."

Fig. 9

Expression of HcNCED3 at different times under salt (A) or drought (B) stress * and ** indicate significantly different at the 0.05 and 0.01 probability levels, respectively. Error bars represent the standard error of each treating group (n = 3)."

Fig. 10

Expression of HcTPPJ at different times under ABA stress Error bars represent the standard error of each treating group (n = 3)."

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