| Soybean[Glycine max(L)Merr.]is an important oil crop in the world and also plays an important role in the adjustment of planting structure in China,and its seeds contain abundant plant protein as well.Spring soybean seeds often encounter the hot and rainy summer during physiological maturation.Seed deterioration occurs in the filed before harvesting and seriously restricts the yield and quality of spring soybean in southern China.Upstream regulatory networks of gene expression play an important role in plant growth and development,biotic and abiotic stresses.Previous studies in our laboratory showed that the soybean gene GmSBH1 was strongly responsive to high temperature and humidity(HTH)stress,and transgenic plants could significantly improve the resistance to HTH.According to the previous research in our laboratory,we carried out the following research:(1)Separation of GmSBH1 promoter and identification of core cis-elements in HTH stress,(2)Seeds of pre-harvest seed deterioration-resistant cv.Xiangdou No.3 treated with HTH stress were used as materials to construct the HTH yeast one-hybrid cDNA library,(3)We used HTH cis-acting element HSE as bait to screen yeast one-hybrid cDNA library,(4)Validation of cis-acting element HSE combined with soybean transcription factor GmCOL4,(5)Interaction between GmCOL4 and GmZTL1 was validated in vitro and in vivo by GST pull-down,bimolecular fluorescence complementation(BiFC)and yeast two-hybrid technique,respectively.(6)The role of soybean genes GmCOL4 and GmZTLl in plant morphogenesis and seed deterioration resistance was elucidated by fluorescence quantitative analysis,functional verification of transgenic plants and subcellular localization techniques,etc.All studies aim to provide a theoretical basis for clarifying the upstream regulatory network of GmSHB1 involving in seed field deterioration.The main results were as follows:1.The GmSBH1 promoter(GmSBH1P,2040 bp)was isolated from the soybean genome DNA,and the GmSBH1P contains a variety of cis-acting elements,according to the promoter online prediction software.Typical TATA box and CAAT box,two types of heat-responsive elements(HSE,STRE),and many other cis-acting elements of responsive to biotic and abiotic stresses were detected on the GmSBH1P.The results of GmSBH1P 5'-terminal deletions revealed that the fragment from-1121 to-638 bp was necessary for the promoter activity in respond to HTH stress.The mutation and deletion of HSE indicates that HSE is the core cis-element in responding to high temperature and humidity.2.In our study,the quality of the yeast one-hybrid cDNA library is good,and the transformation efficiency of the library is higher than 1×106.These results suggested that the yeast one-hybrid cDNA library was well-suited to large-scale screen DNA-protein interactions for us.We used HTH cis-acting element HSE as bait to screen yeast one-hybrid cDNA library.169 positive clones were screened could be interaction with HSE by growth on a nutrient deficient medium.We extracted the plasmid from these 169 positive clones,and used primers to amplify these plasmids by PCR.We found the 169 positive clones belong to 82 different proteins.According to UniProt protein database and NCBI database,the functions of the 82 proteins can be roughly divided into 8 categories:storage protein,seed mature protein,defense protein,ribosomal protein,signal transduction protein,enzyme and protease inhibitors,other functional proteins and unknown functional proteins.3.Plant development and stress related protein,GmCOL4,and its full ORF(930 bp)was isolated from soybean leaves.GmCOL4 encoded a peptide of 310 amino acids.Protein sequence analysis showed that GmCOL4 belongs to the zinc finger transcription factor BBX family.Protein domain analysis showed that GmCOL4 has 3 conserved domains:BBOX1,BBOX 2 and CCT.GmCOL4-GFP fusion protein was only found in the nucleus in N.benthamiana epidermis cells.GmCOL4 had transcriptional activation fuuction,and transcriptional activation sites located in the 51-253 aa which contains BBOX2 domain.Interestingly,the BBOX1 domain was found to play an obvious role in suppressing transcriptional activation activity of GmCOL4.Inserting three tandem repeats of various cis-DNA elements into the pHis2 showed that 3×HSE element shows stronger binding ability with GmCOL4 than the others.4.According to previous studies and software analysis,GmCOL4 may interact with GmZTL1.Full ORF of GmZTL1(1581 bp)was isolated from soybean leaves.GmZTL1 encoded a peptide of 617 amino acids.Protein sequence analysis showed that GmZTLl belonged to F-BOX family.Protein domain analysis showed that GmZTL1 has 3 conserved domains:PAS domain,F-BOX and Kelch domain.GmZTL1-GFP fusion protein was only found in the nucleus in N.benthamiana epidermis cells.Interaction between GmCOL4 and GmZZTL1 was validated in vitro and in vivo by GST pull-down,bimolecular fluorescence complementation(BiFC)and yeast two-hybrid technique,respectively.5.In the present study,our results also indicated that GmCOL4 and GmZTL1 were showed diverse expression patterns among root,stem,leaf,flower,cotyledon,pod and seed in the pre-harvest seed deterioration-sensitive soybean cv.Ningzhen No.1 and-resistant cv.Xiangdou No.3.As far as cv.Ningzhen No.1 is concerned,GmCOL4 was expressed in higher level in mature seed than in the other tissues,but in cv.Xiangdou No.3,GmCOL4 was expressed in higher level in cotyledon and mature pod than in the other tissues.In cv.Ningzhen No.1 and cv.Xiangdou No.3,GmZTL1 was expressed in higher level in mature pod and mature seed than in the other tissues,and increased a little during pod and seed development compared to the control.To determine whether GmCOL4 and GmZTL1 were involved in response to HTH stress or not,its expression level was detected in R7 seeds of soybean cv.Ningzhen No.1 and Xiangdou No.3 treated under HTH stress for 0,6,12,24.48,96 and 168 h.In cv.Ningzhen No.1,the expression levels of GmCOL4 were found to be higher under HTH stress at 12 and 96 h than the control,and in cv.Xiangdou No.3,the expression levels of GmCOL4 were found to be higher under HTH stress at 6.12,24 and 168 h.We found that the trend of GmZTL1 was similar to that of GmCOL4,but in cv.Xiangdou No.3,GmZTL1 was down-regulated in the developing seeds of cv.Ningzhen No.1 under HTH stress 48 h compared to the control.These results suggested that GmCOL4 and GmZTL1 in the developing seeds of soybean might be also involved in response to HTH stress.6.Overexpression of GmZTL1 in the leaves of Nicotiana benthamiana could whiten the leaves of Nicotiana benthamiana,but it did not affect the normal growth of the Nicotiana benthamiana.Transmission electron microscope analysis showed that in transgenic lines of GmZTL1,GmOL4 and the wild type,a large amount of starch grains were accumulated in their mesophyll cells,and chloroplasts were seriously squeezed.These phenomena were not found in albino leaves of GmZTL1,but we found that the contour of albino leaves of GmZTL1 cells became irregular.Analysis of the chloroplast structure showed that showed that in the GmCOL4 transgenic lines,granal lamellae(GL)was closely distributed and stroma lamella(SL)was distorted,and in GmZTL1 transgenic lines,a large number of osmium particles(OP)appeared in albino leaves of GmZTL1.The paraffin section method was used for abnormal structure of leaf at the cellular level.Compared to the wild type,GmCOL4 transgenic lines have a large number of closely arranged palisade cells,meanwhile,in GmZTL1 transgenic lines,palisade cells turned to be short and irregular(normal leaves of GmZTL1),both adaxial and abaxial epidermic cells have become bigger,in addition,albino leaves reduce a large number of palisade cells and sponge cells(albino leaves of GmZTL1).Seed germination rate vigor of GmCOL4 transgenic plants were significantly higher than wild-tyle,and GmZTL1 transgenic plants showed the similar to GmCOL4 transgenic plants.The above results showed that GmCOL4 and GmZTL1 are played an important role in plant growth and development and in response to HTH stress. |
PDF Full Text Request