Functional Analysis Of Soybean GmGBP1Gene Involved In Photoperiodic Flowering Pathway And Stress Response

Soybean is not only an important food crop and oil crop, but also humanity’s main source ofedible protein and industrial raw materials. According to the statistics of National Grain and OilInformation Center, soybean acreage in china was6.75million hectares in2012, a decrease of14.43%over the previous year, and soybean production in2012is expected to be12.8million tons,a decrease of11.63%over the14.49million tons of the previous year. The soybean imports ofchina in2012reached a new record high to58.38million tons, and was a big increase of10.92%compared with52.63million tons in2011. The dependence on foreign soybean of our country in2012was further improved to82%compared with78.4%of2011. The less competation ofsoybean in our country is closely related to the poorer quality, more unstably sustainedproductivity, and lower yield.Wide adaptability, abiotic stresses and other factors have been major problems in China’ssoybean production. The high sensitivity to photoperiod of soybean growth and developmentseriously affects the spanned planting, adaptability and yield of soybean varieties. Biotic andabiotic stresses are also important limiting factors that affect plant growth and development.Abiotic stresses such as heat, cold, drought and salinity stress seriously affect the growth of crops,and result in significant reduction of the crop. The abiotic stresses lead to a decrease of soybeanproduction by30%to50%, and affected the yield and quality of sobean seriously every year inAsia. Therefore, to solve the passive situation of the low soybean productivity fundamentaly, thefirst key method is breaking the limitations of the soybean-growing areas by altering the soybeansensitivity to light to improve the adaptability of soybean varieties, expand introduction of sobeanvarieties, make soybean flower and mature normaly in different regions. While the another keyway is ensuring that soybean yield could be high and stable under different environments byenhancing soybean resistance to stresses, and improve the environmental adaptability of aoybeanin order to expand the acreage of soybeans.SKIP is a transcription cofactor in manyall eukaryotes. All the SKIP homologs identified sofar contained a SNW/SKIP domain with an S-N-W-K-N peptide signature and may have conservedbasic functions, such as acting as a cofactor in transcription and splicing. The study of SKIP inArabidopsis and rice revealed that the expression of SKIP was reduced by a variety of hormonesand stresses. SKIP played a positive role in the regulation of stresses, such as the high salt anddrought stress. SKIP could regulate cell viability and maintain the noraml growth and development of plant. SKIP also involved in plant flowering and regulated the flowering time.To obtain a new soybean gene with the composite functions of low sensitivity to photoperiodand improved stress resistance, in this study, Real-time RT-PCR was used to analyze the spatialand temporal expression pattern of soybean GAMYB binding protein (GmGBP1) gene encoding aSKIP homolog protein, and the expression patterns of GmGBP1of soybean treated with differentday-lengths, stresses and exogenous hormones. The yeast two-hybrid was performed to identify theprotein interactions between GmGBP1and GmGAMYB1. GmGBP1was over-expression in themodel plant Arabidopsis thaliana and Arabidopsis myb33mutant. RNAi method was used tosuppress the Arabidopsis endogenous SKIP gene. the phenotype and physiological activity oftransgenic lines were analyzed to identify the function of GmGBP1in plant. The main results wereas follows:1. Soyben has two copies of SKIP gene named GmGBP1and GmGBP2respectively. GmGBP1is located in chromosome16and GmGBP2is located in chromosome1. The the amino acidhomology rate between GmGBP1and GmGBP2is96.73%. The full length cDNA of GmGBP1,containing2253bp with an open reading frame of1,839bp, was predicted to encode612aminoacids. GmGBP1protein had a predicted isoelectric point (pI) of8.69and a molecular weight (MW)of69.10kD, was a hydrophilic protein. GmGBP1has a SKIP/SNW protein domain locatedbetween amino acids190and360with an S-N-W-K-N peptide signature.2. Real-time RT-PCR analysis showed that GmGBP1could express in soybean root, leaf,trifoliate leaf, stem, flower bud, pod and immature embryo. In long-day conditions (LDs), theexpression levels of GmGBP1were much higher in immature embryo, stem and root. In short-dayconditions (SDs), the expression levels of GmGBP1were much higher in trifoliate leaf, immatureembryo and flower bud. All the organs, except of stem, showed higher expression of GmGBP1inSDs than in LDs.3. Real-time RT-PCR analysis showed that GmGBP1was induced by GA3and ABA. Theexpression of GmGBP1in soybean was up-regulated, up to peak at2h after treated with100μmolGA3and then up-regulated continuously. The expression of GmGBP1in soybean was up-regulated,and up to peak at1h after treated with100μmol ABA.4. Real-time RT-PCR analysis showed that GmGBP1was induced by abiotic stresses. Theexpression of GmGBP1in soybean was up-regulated by PEG6000, up to peak at4h after treatedwith8%PEG6000. The expression of GmGBP1in soybean was up-regulated by NaCl, and up topeak at4h after treated with200mM NaCl. The expression of GmGBP1in soybean wasup-regulated by heat, and up to peak at2h after treated with42°C.5. Prokaryotic expression vector pGEX-6p-GmGBP1was constructed and transformed intoE.coli BL21. The recombinant GmGBP1proteins was95kD and expressed in inclusion bodies.6. Yeast expression vectors pBD-GmGBP1with deletion mutants of GmGBP1wereconstructed and transformed into yeast. It showed that GmGBP1had the transcriptional activity in its C-terminal domain. Yeast expression vectors pAD-GmGAMYB1with deletion mutants ofGmGAMYB1were constructed and co-transformed with pBD-GmGBP1into yeast. Yeasttwo-hybrid analysis showed that the SKIP domain of GmGBP1could interact with the N-terminaldomain of GmGAMYB1.7. The pCAMBIA3300-pBI121-GmGBP1vector was used to transforme Arabidopsis andArabidopsis myb33mutant respectively. The transgenic plants were screened and identified by PPT,PCR and RT-PCR, resulting in3homozygous lines of T3transgenic Arabidopsis withover-expression of GmGBP1(GmGBP1-ox) and3homozygous lines of T3transgenic Arabidopsismyb33mutant with over-expression of GmGBP1(GmGBP1-ox/myb33). The pJawoh18-GmGBP1vector was used to suppress the Arabidopsis endogenous SKIP gene (atskip-i). The transgenicplants were screened and identified by PPT, PCR and RT-PCR., resulting in3homozygous lines ofT3transgenic Arabidopsis with suppression of AtSKIP.8. In LDs, GmGBP1-ox plants flowered earlier than wild-type (WT) plants,GmGBP1-ox/myb33plants flowered as same as WT, atskip-i plants flowered later than WT plants.RT-PCR analysis showed that GmGBP1up-regulated the expression of CONSTANS (CO) andFLOWERING LOCUS T (FT) to promote flowering, while GmGBP1up-regulated the expressionof LFY by binding with MYB33to promote flowering. In SDs, GmGBP1-ox andGmGBP1-ox/myb33plants flowered both later than WT plants, atskip-i plants flowered earlier thanWT plants. RT-PCR analysis showed that GmGBP1up-regulated the expression of FLOWERINGLOCUS C (FLC) to delay flowering.9. With GA3treatment, the seed germination of GmGBP1-ox plants was promoted signally,and the promotion of atskip-i plants was less than GmGBP-ox plants. With PAC treatment, the seedgermination of GmGBP1-ox plants was inhibited obviously, and the inhibition of atskip-i plantswas less than GmGBP1-ox plants. With ABA treatment, the seed germination of GmGBP1-ox andatskip-i plants were both inhibited.10. Phenotype, the contents of MDA and SOD, the expression of stress-related genes wereanalyzed after plants treated with NaCl, drought and heat respectively. It showed that GmGBP1reduced the tolerance of NaCl stress and enhanced the resistance to drought and heat stress.