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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (4): 1017-1026.doi: 10.3724/SP.J.1006.2022.04274

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Cloning and functional analysis of ScGA20ox1 gibberellin oxidase gene in sugarcane

ZHOU Hui-Wen1(), QIU Li-Hang1, HUANG Xing, LI Qiang2, CHEN Rong-Fa1, FAN Ye-Geng1, LUO Han-Min1, YAN Hai-Feng1, WENG Meng-Ling1, ZHOU Zhong-Feng1,*(), WU Jian-Ming1,*()   

  1. 1Key Laboratory of Sugarcane Biotechnology and Genetic Improvement, Ministry of Agriculture and Rural Affairs / Guangxi Key Laboratory of Sugarcane Genetic Improvement, Sugarcane Research Institute / Guangxi Zhuang Autonomous Region Academy of Agri-cultural Sciences, Nanning 530007, Guangxi, China
    2Scientific Research Institute of Zhanjiang State Farms, Zhanjiang 524086, Guangdong, China
  • Received:2021-04-07 Accepted:2021-09-09 Online:2022-04-12 Published:2021-10-18
  • Contact: ZHOU Zhong-Feng,WU Jian-Ming E-mail:windyrentmd@qq.com;445708586@qq.com;wujianming2004@126.com
  • Supported by:
    Sub-project of the National Key Research and Development Program of China(2019YFD1000503);Natural Science Foundation of Guangxi, China(2021GXNSFAA220014);Guangxi Innovation Team of China Agricultural Research System(nycytxgxcxtd-2021-03-02)

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Abstract:

GA20-oxidase (GA20ox) is a key rate-limiting enzyme in the synthesis of gibberellic acid (GA), but the biological function and expression pattern of gene GA20ox1 in sugarcane remain unknown. In this study, we cloned GA20-oxidase gene (ScGA20ox1) from the sugarcane variety ROC22 sequence by RT-PCR and RACE, which had a total length of 1574 bp and a complete open reading frame (ORF) of 1125 bp that encoded 375 amino acids. The ScGA20ox1 protein is a hydrophilic protein with a molecular weight of 42.3 kD, an iscelectric point (pI) of 5.95, and no transmembrane structure or signal peptide. Real-time fluorescence quantitative analysis revealed that ScGA20ox1 had the highest expression levels in stems, medium in the leaves, and the lowest in roots of sugarcane seedlings. Drought stress, low temperature stress, and GA3 treatment change the expression patterns of ScGA20ox1 in different tissues. ScGA20ox1 was transformed into Arabidopsis via Agrobacterium-mediated transformation. Transgenic Arabidopsis thaliana plants with overexpressed ScGA20ox1 were obtained from the transformed Arabidopsis thaliana via Agrobacterium-mediated transformation, which showed a phenotypic variation of plant height and internode length increase. In conclusion, ScGA20ox1 was involved in sugarcane’s growth as a crucial regulator in response to abiotic stress and the phenotypic variation of overexpressed transgenic Arabidopsis thaliana changed, which would lay a theoretical basis for future in-depth studies on ScGA20ox1’s biological function and analysis of the molecular mechanism of sugarcane plant shape regulation.

Key words: sugarcane, gibberellin oxidase, gene cloning, expression level

Table 1

Primers used in cloning and expression analysis of ScGA20ox1"

引物
Primer		 引物序列
Primer sequences (5'-3')		 用途
Function
F1 CATCCCGC(T)A(C)GCAGTTCATCT 中间片段 Intermediate fragment
R1 CAACATCGGCGACACCTTCAT
5R1 CCTCAACTACTACCCGCCGT 5' RACE
5R2 ACTTCCGCCGCTTCTTCCAG
3F1 TTCTTCACGCTCCCGCTGTC 3' RACE
3F2 ACTTCCGCCGCTTCTTCCAG
F2 ATGGTGCAGCAGGCTGCGC 编码区 Coding region
R2 GGCCTCATCACCTCCATGA
GA20-qF CGGCTTCTTCCAGGTGGTCAA RT-qPCR
GA20-qR TGGAAGGAGACGCTGTCGTTC
25S rRNA-qF GCAGCCAAGCGTTCATAGC RT-qPCR
25S rRNA-qR GGATTGTTCACCCACCAATAGG
knpGA20-F GGGGTACCATGGTGCAGCAGGCTGCGC 载体构建 Vector construction
bamGA20-R CGGGATCCTCATGGAGGTGATGAGGCC 载体构建 Vector construction

Fig. 1

Agarose gel electrophoresis of PCR product for ScGA20ox1 cloning M: DL2000 marker; A: amplification result of ScGA20ox1 fragment; B: amplificatin result of 3' RACE; C: amplificatin result of 5' RACE; D: amplificatin result of ORF."

Fig. 2

Conserved functional domain of ScGA20ox1"

Fig. 3

Predicted 3-D structure of ScGA20ox1"

Fig. 4

Multiple sequence alignment analysis of GA20ox in different plants CBX45610: Triticum aestivum; NP 001241783: Zea mays; AAB48239: Oryza sativa; AAT49058: Hordeum vulgare; KY416517: Saccharum officinarum; XP-002463483: Sorghum bicolor. In the black box there are the LPWKET and NYYPPCQRP conserved domains in turn."

Fig. 5

Phylogenetic relationship of amino acid sequences between ScGA20ox1 and other GA20ox proteins"

Fig. 6

Different expression of ScGA20ox gene in different organ and different treatment A: different organ; B: drought stress; C: low temperature stress; D: GA treatment. Different lowercase letters indicate significant differences between the expression level from the same tissue at 0, 24, 48 hour(s) after treatment at P < 0.05."

Fig. 7

PCR detection of resistant plants M: DL2000 marker; 1: the blank with deionized water; 2:pEASY-ScGA20ox1vector; 3: wild-type Arabidopsis thaliana; 4-13: transgenic Arabidopsis thaliana."

Fig. 8

Expression value of ScGA20ox1 in wild type (WT) and transgenic Arabidopsis thaliana (ga20-1-ga20-10) strain Different lowercase letters indicate significant differences between the treatments at P < 0.05."

Fig. 9

Determination of phenotype in over-expression transgenic Arabidopsis thaliana A: the phenotype observation; B: the measurement of plant height; C: the measurement of internodes length; D: the measurement of numbers of internodes. WT: wild-type plants of Arabidopsis thaliana; ga20-3, ga20-5, and ga20-6 represent transgenic plants of Arabidopsis thaliana. Different lowercase letters indicate significant differences between the treatments at P < 0.05."

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