Cloning and Expression of GA20ox, NCED and IAA and Their Roles in Bulb Development of Fritillaria thunbergii
LI Zi-Ming1, FAN Xiao-Ping1, JIN Ze-Lan1, JIANG Jian-Ming2, WANG Zhi-An2, WANG Zhong-Hua1,*
1 Institute of Biotechnology, Zhejiang Wanli University, Ningbo 315100, China; 2 Zhejiang Institute of Traditional Chinese Medicine, Hangzhou 310023, China
Abstract Gibberellin (GA) is a type of diterpene plant hormone, which plays an important role in the physiological development of plants. GA can promote the plant extension and affect the production of dry matter in sink organs. It can also activate α-amylase activity to promote catalyzes the degradation of energy storage substances and promotes the break of plant dormancy. For example, the increase of GA content can promote the germination of barley and make it enter the development stage earlier. GA also has an effect on fruit development and maturity, it can also delay leaf senescence and inhibit tuber formation. Gibberellin 20- oxidase (GA20ox) enzyme is the key rate-limiting enzyme in the final stage of GA synthesis pathway. It exercises catalytic regulation function in the final stage of GA synthesis and promotes the production of GA20 and GA9, those are precursors of active GA1 and GA4. GA20ox gene has a close influence on the activity of GA20ox enzyme and is closely related to GA. ABA (abscisic acid) is a 15-carbon sesquienene compound, which can effectively promote the development and growth of various plants. It plays an important role in seed germination, maturation and dormancy, fruit maturation. It can bring huge economic benefits and can effectively increase crop yields. There are 2 ways to synthesize ABA in plants, one is the indirect pathway, the C15 pathway, and the other is the direct pathway, which also can be called the carotenoid pathway. The synthesis of ABA in higher plant tissues is mainly achieved through indirect pathways. 9-cis epoxy carotenoid dioxygenase (NCED) is the rate-limiting enzyme in the process of indirect ABA synthesis and regulation. Auxin (IAA) is an endogenous hormone containing an unsaturated aromatic ring and an acetic acid side chain. IAA is a signal transduction compound that promotes and affects plant development and physiological changes. It plays an important role at every moment of development, affecting the division, differentiation and elongation of plant cells, as well as various physiological activities of plants, such as apical dominance, light orientation and gravity, and induces many division-related genes expression. Indoleacetic acid (AUX)/IAA auxin response protein can negatively regulate auxin synthesis. In this study, enzyme linked ELISA was used to determine the changes in GA, ABA, and IAA content in Fritillaria thunbergii during bulb development. As the bulb develops, GA content showed a downward trend. ABA showed an upward trend, IAA generally showed a downward trend. Using homologous cloning combined and RACE technology, Fritillaria thunbergii GA20ox (GenBank No. MW238816), NCED (GenBank No. MW238817) and IAA (GenBank No. MW238818) were successfully cloned. Bioinformatics analysis showed that the full length of the sequence of GA20ox gene was 1 490 bp, the open reading frame was 1 122 bp, and encoded 373 amino acids; the full length of the sequence of NCED gene was 2 270 bp , and the open reading frame was 1 800 bp, which encoded 599 amino acids; the full length of the sequence of IAA gene was 1 255 bp, with an open reading frame of 864 bp, encoding 287 amino acids. qRT-PCR analysis showed that the GA20ox, NCED, IAA genes expression in bulbs at different developmental stages correlated with GA, ABA, and IAA. The correlation analysis showed that GA20ox, NCED and GA, ABA content were significantly positively correlated (P<0.01), IAA and IAA content was a very significant negative correlation (P<0.01). This study provides a theoretical basis for the study of the functions of GA20ox, NCED, IAA genes in GA, ABA and IAA metabolism, and provides references for further exploring the molecular mechanisms of GA20ox, NCED and IAA genes in the bulb development process of F. thunbergii.
LI Zi-Ming,FAN Xiao-Ping,JIN Ze-Lan, et al. Cloning and Expression of GA20ox, NCED and IAA and Their Roles in Bulb Development of Fritillaria thunbergii[J]. 农业生物技术学报, 2021, 29(9): 1710-1721.
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