1.Functional analysis on sucrose transporters in sweet potato.
Yiran LIU ; Zhengdan WU ; Weitai WU ; Chaobin YANG ; Cairui CHEN ; Kai ZHANG
Chinese Journal of Biotechnology 2023;39(7):2772-2793
		                        		
		                        			
		                        			Sweet potato is an important food crop that can also be used as an industrial raw material. Sucrose is the main form of long-distance carbohydrate transport in plants, and sucrose transporter (SUT) regulates the transmembrane transport and distribution of sucrose during plant growth and metabolism. Moreover, SUT plays a key role in phloem mediated source-to-sink sucrose transport and physiological activities, supplying sucrose for the sink tissues. In this study, the full-length cDNA sequences of IbSUT62788 and IbSUT81616 were obtained by rapid amplification of cDNA ends (RACE) cloning according to the transcripts of the two SUT coding genes which were differentially expressed in sweet potato storage roots with different starch properties. Phylogenetic analysis was performed to clarify the classification of IbSUT62788 and IbSUT81616. The subcellular localization of IbSUT62788 and IbSUT81616 was determined by transient expression in Nicotiana benthamiana. The function of IbSUT62788 and IbSUT81616 in sucrose and hexose absorption and transport was identified using yeast functional complementarity system. The expression pattern of IbSUT62788 and IbSUT81616 in sweet potato organs were analyzed by real-time fluorescence quantitative PCR (RT-qPCR). Arabidopsis plants heterologous expressing IbSUT62788 and IbSUT81616 genes were obtained using floral dip method. The differences in starch and sugar contents between transgenic and wild-type Arabidopsis were compared. The results showed IbSUT62788 and IbSUT81616 encoded SUT proteins with a length of 505 and 521 amino acids, respectively, and both proteins belonged to the SUT1 subfamily. IbSUT62788 and IbSUT81616 were located in the cell membrane and were able to transport sucrose, glucose and fructose in the yeast system. In addition, IbSUT62788 was also able to transport mannose. The expression of IbSUT62788 was higher in leaves, lateral branches and main stems, and the expression of IbSUT81616 was higher in lateral branches, stems and storage roots. After IbSUT62788 and IbSUT81616 were heterologously expressed in Arabidopsis, the plants grew normally, but the biomass increased. The heterologous expression of IbSUT62788 increased the soluble sugar content, leaf size and 1 000-seed weight of Arabidopsis plants. Heterologous expression of IbSUT81616 increased starch accumulation in leaves and root tips and 1 000-seed weight of seeds, but decreased soluble sugar content. The results obtained in this study showed that IbSUT62788 and IbSUT81616 might be important genes regulating sucrose and sugar content traits in sweet potato. They might carry out physiological functions on cell membrane, such as transmembrane transport of sucrose, sucrose into and out of sink tissue, as well as transport and unloading of sucrose into phloem. The changes in traits result from their heterologous expression in Arabidopsis indicates their potential in improving the yield of other plants or crops. The results obtained in this study provide important information for revealing the functions of IbSUT62788 and IbSUT81616 in starch and glucose metabolism and formation mechanism of important quality traits in sweet potato.
		                        		
		                        		
		                        		
		                        			Ipomoea batatas/metabolism*
		                        			;
		                        		
		                        			Arabidopsis/metabolism*
		                        			;
		                        		
		                        			Sucrose/metabolism*
		                        			;
		                        		
		                        			Saccharomyces cerevisiae/metabolism*
		                        			;
		                        		
		                        			DNA, Complementary
		                        			;
		                        		
		                        			Phylogeny
		                        			;
		                        		
		                        			Plants, Genetically Modified/genetics*
		                        			;
		                        		
		                        			Membrane Transport Proteins/metabolism*
		                        			;
		                        		
		                        			Starch/metabolism*
		                        			;
		                        		
		                        			Plant Proteins/metabolism*
		                        			;
		                        		
		                        			Gene Expression Regulation, Plant
		                        			
		                        		
		                        	
2.Genome-wide identification of SUN gene family in Fragaria vesca and stresses-response analysis.
Yao YU ; Ziyao WANG ; Yiling XU ; Bojun MA ; Xifeng CHEN
Chinese Journal of Biotechnology 2023;39(2):724-740
		                        		
		                        			
		                        			SUN gene is a group of key genes regulating plant growth and development. Here, SUN gene families of strawberry were identified from the genome of the diploid Fragaria vesca, and their physicochemical properties, genes structure, evolution and genes expression were also analyzed. Our results showed that there were thirty-one FvSUN genes in F. vesca and the FvSUNs encoded proteins were classified into seven groups, and the members in the same group showed high similarity in gene structures and conservative motifs. The electronic subcellular localization of FvSUNs was mainly in the nucleus. Collinearity analysis showed that the members of FvSUN gene family were mainly expanded by segmental duplication in F. vesca, and Arabidopsis and F. vesca shared twenty-three pairs of orthologous SUN genes. According to the expression pattern in different tissues shown by the transcriptome data of F. vesca, the FvSUNs gene can be divided into three types: (1) expressed in nearly all tissues, (2) hardly expressed in any tissues, and (3) expressed in special tissues. The gene expression pattern of FvSUNs was further verified by quantitative real-time polymerase chain reaction (qRT-PCR). Additionally, the seedlings of F. vesca were treated by different abiotic stresses, and the expression level of 31 FvSUNs genes were assayed by qRT-PCR. The expression of most of the tested genes was induced by cold, high salt or drought stress. Our studies may facilitate revealing the biological function and molecular mechanism of SUN genes in strawberry.
		                        		
		                        		
		                        		
		                        			Fragaria/metabolism*
		                        			;
		                        		
		                        			Genes, Plant
		                        			;
		                        		
		                        			Stress, Physiological/genetics*
		                        			;
		                        		
		                        			Arabidopsis/genetics*
		                        			;
		                        		
		                        			Plant Development
		                        			;
		                        		
		                        			Gene Expression Regulation, Plant
		                        			;
		                        		
		                        			Plant Proteins/metabolism*
		                        			
		                        		
		                        	
3.Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro.
Ke-Ke ZHANG ; Shu-Fu SUN ; Yu-Ping TAN ; Zhao-Yang XU ; Yin-Yin JIANG ; Jian YANG ; Da-Yong LI ; Jin-Fu TANG
China Journal of Chinese Materia Medica 2023;48(6):1510-1517
		                        		
		                        			
		                        			Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.
		                        		
		                        		
		                        		
		                        			Isatis/genetics*
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		                        			Plant Proteins/metabolism*
		                        			;
		                        		
		                        			Phylogeny
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		                        			Arabidopsis/genetics*
		                        			;
		                        		
		                        			Flavonoids
		                        			;
		                        		
		                        			Cloning, Molecular
		                        			
		                        		
		                        	
4.Expression of Brassica juncea BjuWRKY75 and its interactions with flowering integrator BjuFT.
Junjie FENG ; Yuanda WANG ; Qinlin DENG ; Haitao ZHAI ; Xu YE ; Dayong WEI ; Zhimin WANG ; Qinglin TANG
Chinese Journal of Biotechnology 2022;38(8):3029-3040
		                        		
		                        			
		                        			Brassica juncea is a yearly or biennial vegetable in Brassica of Cruciferae. The yield and quality of its product organs are affected by flowering time. WRKY proteins family can respond to biological and abiotic stresses, developmental regulation and signal transduction. WRKY75 is an important member of WRKY family which can regulate flowering, but the flowering regulation mechanism in B. juncea has not been reported. In this study, a gene BjuWRKY75 in B. juncea was cloned, and the encoded-protein belonged to the group Ⅱ of WRKY protein with highly conserved domain. BjuWRKY75 had the highest homology with BriWRKY75 of Brassica nigra. The relative expression level of BjuWRKY75 in flowers was significantly higher than that in leaves and stems, and it was expressed stably in leaves. BjuWRKY75 protein was localized in the nucleus and interacted with the promoter of the flowering integrator BjuFT, which contained the W-box response element for the interaction between protein and DNA. Thus, it could transcriptionally activate the expression of the downstream genes. The overexpression of BjuWRKY75 in Arabidopsis led to earlier flowering significantly. In conclusion, BjuWRKY75 could directly target the promoter of BjuFT and accelerate flowering. These results may facilitate further study on the regulation of flowering molecules of BjuWRKY75.
		                        		
		                        		
		                        		
		                        			Arabidopsis/genetics*
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		                        			Flowers/genetics*
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		                        			Gene Expression Regulation, Plant
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		                        			Mustard Plant/genetics*
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		                        			Plant Proteins/metabolism*
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		                        			Promoter Regions, Genetic
		                        			
		                        		
		                        	
5.The role of SnRK2 in the response to stress, the growth and development of plants.
Zixi LIU ; Yaxin ZHU ; Guoqiang WU ; Ming WEI
Chinese Journal of Biotechnology 2022;38(1):89-103
		                        		
		                        			
		                        			Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) is a specific Ser/Thr protein kinase in plants. SnRK2 can regulate the expression of downstream genes or transcription factors through phosphorylation of substrates to achieve stress resistance regulation in different tissue parts, and make plants adapt to adverse environment. SnRK2 has a small number of members and a molecular weight of about 40 kDa, and contains a conserved N-terminal kinase domain and a divergent C-terminal regulatory domain, which plays an important role in the expression of enzyme. This review summarized the recent research progresses on the discovery, structure, and classification of SnRK2, and its function in response to various stresses and in regulating growth and development, followed by prospecting the future research direction of SnRK2. This review may provide a reference for genetic improvement of crop stress resistance.
		                        		
		                        		
		                        		
		                        			Abscisic Acid
		                        			;
		                        		
		                        			Arabidopsis Proteins/genetics*
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		                        			Gene Expression Regulation, Plant
		                        			;
		                        		
		                        			Growth and Development
		                        			;
		                        		
		                        			Plants/genetics*
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		                        			Protein Kinases
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		                        			Protein Serine-Threonine Kinases/genetics*
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		                        			Stress, Physiological/genetics*
		                        			
		                        		
		                        	
6.Mechanisms of alternative splicing in regulating plant flowering: a review.
Huanhuan LU ; Qinlin DENG ; Mengdan WU ; Zhimin WANG ; Dayong WEI ; Hebing WANG ; Huafeng XIANG ; Hongcheng ZHANG ; Qinglin TANG
Chinese Journal of Biotechnology 2021;37(9):2991-3004
		                        		
		                        			
		                        			Flowering is a critical transitional stage during plant growth and development, and is closely related to seed production and crop yield. The flowering transition is regulated by complex genetic networks, whereas many flowering-related genes generate multiple transcripts through alternative splicing to regulate flowering time. This paper summarizes the molecular mechanisms of alternative splicing in regulating plant flowering from several perspectives, future research directions are also envisioned.
		                        		
		                        		
		                        		
		                        			Alternative Splicing/genetics*
		                        			;
		                        		
		                        			Arabidopsis/metabolism*
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		                        			Arabidopsis Proteins/genetics*
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		                        			Flowers/genetics*
		                        			
		                        		
		                        	
7.Ectopic expression of the AmDREB1F gene from Ammopiptanthus mongolicus enhances stress tolerance of transgenic Arabidopsis.
Kuangang TANG ; Bo DONG ; Xiaojun WEN ; Yumei YIN ; Min XUE ; Zixian SU ; Maoyan WANG
Chinese Journal of Biotechnology 2021;37(12):4329-4341
		                        		
		                        			
		                        			Dehydration-responsive element binding proteins (DREBs) are an important class of transcription factors related to plant stress tolerance. Ammopiptanthus mongolicus is an evergreen broadleaf shrub endemic to desert areas of northwest China, and it has a very high tolerance to harsh environments. In order to reveal the functions and mechanisms of the AmDREB1F gene from this species in enduring abiotic stresses, we performed subcellular localization test, expression pattern analysis, and stress tolerance evaluation of transgenic Arabidopsis harboring this gene. The protein encoded by AmDREB1F was localized in the nucleus. In laboratory-cultured A. mongolicus seedlings, the expression of AmDREB1F was induced significantly by cold and drought but very slightly by salt and heat stresses, and undetectable upon ABA treatment. In leaves of naturally growing shrubs in the wild, the expression levels of the AmDREB1F gene were much higher during the late autumn, winter and early spring than in other seasons. Moreover, the expression was abundant in roots and immature pods rather than other organs of the shrubs. Constitutive expression of AmDREB1F in Arabidopsis induced the expression of several DREB-regulated stress-responsive genes and improved the tolerance of transgenic lines to drought, high salinity and low temperature as well as oxidative stress. The constitutive expression also caused growth retardation of the transgenics, which could be eliminated by the application of gibberellin 3. Stress-inducible expression of AmDREB1F also enhanced the tolerance of transgenic Arabidopsis to all of the four stresses mentioned above, without affecting its growth and development. These results suggest that AmDREB1F gene may play positive regulatory roles in response to abiotic stresses through the ABA-independent signaling pathways.
		                        		
		                        		
		                        		
		                        			Arabidopsis/metabolism*
		                        			;
		                        		
		                        			Droughts
		                        			;
		                        		
		                        			Ectopic Gene Expression
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		                        			Fabaceae/genetics*
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		                        			Gene Expression Regulation, Plant
		                        			;
		                        		
		                        			Plant Proteins/metabolism*
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		                        			Plants, Genetically Modified/genetics*
		                        			;
		                        		
		                        			Stress, Physiological/genetics*
		                        			
		                        		
		                        	
8.Cloning and functional analysis of AaHSFB1 and its promoter in Amorphophallus.
Yinghong WANG ; Bei ZHANG ; Nan ZHANG ; Zhenyu YUE ; Zhimin WANG ; Shenglin ZHANG ; Yi NIU
Chinese Journal of Biotechnology 2021;37(12):4351-4362
		                        		
		                        			
		                        			To explore the function of a heat shock transcription factor gene (HSFB1) and its promoter in Amorphophallus, a 1 365 bp DNA sequence was obtained by homologous cloning from Amorphophallus albus. The gene expression level of AaHSFB1 determined by qRT-PCR indicated that AaHSFB1 gene is more sensitive to heat stress. The expression level of AaHSFB1 in roots increased followed by a decrease upon heat treatment, and the highest expression level was observed after heat treatment for 1 h. The expression level of AaHSFB1 in leaves reached the highest after heat treatment for 12 h. The expression level in bulbs did not change greatly during the heat treatment. Subcellular localization analysis showed that AaHSFB1 protein was localized in the nucleus. A 1 509 bp DNA sequence which contains the AaHSFB1 promoter was obtained by FPNI-PCR method. Bioinformatics analysis showed that the promoter contained heat stress response elements HSE and a plurality of cis-acting elements related to plant development and stress response. A prAaHSFB1::GUS fusion expression vector was constructed to further analyze the function of AaHSFB1 promoter. The expression vector was transformed into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated method, and GUS staining analysis on transgenic plants after heat treatment was performed. The results showed that AaHSFB1 promoter had very high activity in the leaves. Therefore, we speculate that AaHSFB1 may play an important role in the stress resistance of A. albus, especially when encountering heat stress.
		                        		
		                        		
		                        		
		                        			Amorphophallus/metabolism*
		                        			;
		                        		
		                        			Arabidopsis/genetics*
		                        			;
		                        		
		                        			Cloning, Molecular
		                        			;
		                        		
		                        			Gene Expression Regulation, Plant
		                        			;
		                        		
		                        			Plant Proteins/metabolism*
		                        			;
		                        		
		                        			Plants, Genetically Modified/genetics*
		                        			
		                        		
		                        	
9.Heat shock transcription factor family in plants: a review.
Nan ZHANG ; Yinghong WANG ; Zhimin WANG ; Zhenyu YUE ; Yi NIU
Chinese Journal of Biotechnology 2021;37(4):1155-1167
		                        		
		                        			
		                        			With the constant change of global climate, plants are often affected by multiple abiotic stresses such as heat stress, drought stress, cold stress and saline-alkali stress. Heat shock transcription factors (HSFs) are a class of transcription factors widely existing in plants to respond to a variety of abiotic stresses. In this article, we review and summarize the structure, signal regulation mechanism of HSFs and some research in plants like Arabidopsis thaliana, tomato, rice and soybean, to provide reference for further elucidating the role of HSFs in the stress regulation network.
		                        		
		                        		
		                        		
		                        			Arabidopsis/metabolism*
		                        			;
		                        		
		                        			Droughts
		                        			;
		                        		
		                        			Gene Expression Regulation, Plant
		                        			;
		                        		
		                        			Heat Shock Transcription Factors/genetics*
		                        			;
		                        		
		                        			Plant Proteins/genetics*
		                        			;
		                        		
		                        			Stress, Physiological
		                        			;
		                        		
		                        			Transcription Factors/metabolism*
		                        			
		                        		
		                        	
10.Expression and function analysis of FaCO gene in Festuca arundinacea.
Xi CHEN ; Ying CHEN ; Xiaoxia LIU ; Jianhong SHU ; Xiaoli WANG ; Degang ZHAO
Chinese Journal of Biotechnology 2021;37(4):1324-1333
		                        		
		                        			
		                        			Photoperiod plays an important role in transformation from vegetative growth to reproductive growth in plants. CONSTANS (CO), as a unique gene in the photoperiod pathway, responds to changes of day length to initiate flowering in the plant. In this study, the expression level of FaCONSTANS (FaCO) gene under long-day, short-day, continuous light and continuous darkness conditions was analyzed by real-time quantitative PCR. We constructed the over-expression vector p1300-FaCO and infected into Arabidopsis thaliana by Agrobacterium-mediated method. We constructed the silencing vector p1300-FaCO-RNAi and infected into Festuca arundinacea by Agrobacterium-mediated method. The expression of FaCO gene was regulated by photoperiod. The over-expression of FaCO promoted flowering in wild type of Arabidopsis thaliana under long day condition and rescued the late flowering phenotype in co-2 mutant of Arabidopsis thaliana. Silencing FaCO gene in Festuca arundinacea by RNAi showed late-flowering phenotype or always kept in the vegetative growth stage. Our understanding the function of FaCO in flowering regulation will help further understand biological function of this gene in Festuca arundinacea.
		                        		
		                        		
		                        		
		                        			Arabidopsis/metabolism*
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		                        			Arabidopsis Proteins/genetics*
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		                        			Festuca/metabolism*
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		                        			Flowers/genetics*
		                        			;
		                        		
		                        			Gene Expression Regulation, Plant
		                        			;
		                        		
		                        			Photoperiod
		                        			
		                        		
		                        	
            
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