miR156b from Soybean CMS Line Modulates Floral Organ Development

Shouping Yang,Jun-Yi Gai 한국식물학회 2020 Journal of Plant Biology Vol.63 No.2

The miR156 and plant specifc transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) are known for their function regulating plant growth and development. In this study, we identifed 20 GmSPLs which are targeted by gma-miR156b via plant small RNA target and degradome analysis. And we found high transcript levels of gma-miR156b and its targeted GmSPLs in the fower of soybean cytoplasmic male sterility (CMS) line and its maintainer. The gma-miR156b direct cleavage of GmSPL2b and GmSPL9b, and have opposite expression levels during early fower buds development. We observed a high expression level of GUS protein in the anthers of the line with pgma-MIR156b::GUS reporter. Over-expression of the gma-miR156b precursor in Arabidopsis inhibited foral organ development, including reduced anther size and the amount of pollen grains per anther etc. Like miR156-targeted SPL genes, non-targeted GmSPL8s were also down-regulated in early fower bud development of soybean CMS line compared with its maintainer line, which might act in concert with miR156-targeted SPL genes to participate in the foral organ development. Quantitative real time PCR (qRT-PCR) suggested that miR156/SPL modulates foral organ development by regulating the expression of LATERAL ORGAN BOUNDARIES DOMAIN22 (LBD22), LBD36, AGAMOUS-LIKE30 (AGL30) and AGL104. Our fndings will facilitate understanding of the biological functions of miR156/SPL in foral organ development of soybean CMS.

High Efficient in Vitro Shoot Regeneration from Cotyledonary Nodes of Vegetable Soybean

Qian-Qian Liu,Jun-Yi Gai,Yue-Lin Zhu,Gang Chen,Li-Fei Yang,Guo-Ping Wei,Cong Wang 한국원예학회 2008 한국원예학회 기타간행물 Vol.- No.-

Construction and Characterization of Full-Length cDNA Library and Expressed Sequence Tags Analysis in Developing Seeds of Vegetable Soybean

Li-Fei Yang,Jun-Yi Gai,Yue-Lin Zhu,Gang Chen,Guo-Ping Wei,Cong Wang,Qian-Qian Liu 한국원예학회 2009 Horticulture, Environment, and Biotechnology Vol.50 No.1

The objectives of this paper were to construct a full-length cDNA library and identify genes encoding high quality proteins in developing seeds of vegetable soybean [Glycine max (L.) Merr.]. The construction of full-length cDNA library was essential for screening and cloning of novel genes. Using vegetable soybean variety ‘NN99-10’ as an experimental material, a full-length cDNA library from the developing seeds was constructed by switching mechanism at 5’-end of RNA transcript (SMART) approach. Total RNA from different developmental stages of seeds was purified. Double strand cDNA was synthesized by SMART method and the cDNA fragments were ligated to the pBluescript II SK vector. The recombinant plasmids were transformed into E. coli by electroporation and a cDNA library of developing seeds was successfully constructed. By counting the number of colonies, the titer of the primary cDNA library was 7.6×10? cfuㆍ㎖<SUP>-1</SUP>, and that of the amplified library was 2.4×10<SUP>12</SUP> cfuㆍ㎖<SUP>-1</SUP>. Electrophoresis gel results indicated that most of the cDNA inserts were ranged from 0.40 to 2.0 kb, with an average size of larger than 800 bp. Randomly picked clones showed that the recombination rate was above 99%. These evidences showed that the cDNA library constructed was a full-length library with high quality, and could be used to screen the genes encoding high quality proteins. A total of 206 randomly selected positive clones were successfully sequenced, and 73% of them contained full-length coding regions. Based on bioinformatics analysis of the sequences, we obtained 192 expressed sequence tags (ESTs) from 206 cDNA positive clones. Eight important genes which might be involved in seed quality were identified, and two of their genes were identified to be related to the allergenicity of seed proteins. The full-length cDNA library constructed in this research has laid the foundation for the further cloning of novel genes and the revealed EST sequences have enriched our knowledge of the function genes of vegetable soybean seeds.

Construction and Characterization of Full-length cDNA Library and Expressed Sequence Tags Analysis in Developing Seeds of Vegetable Soybean

Li-Fei Yang,Jun-Yi Gai,Yue-Lin Zhu,Gang Chen,Guo-Ping Wei,Cong Wang,Qian-Qian Liu 한국원예학회 2008 한국원예학회 기타간행물 Vol.- No.-

Construction and Characterization of a Full-length cDNA Library and Identification of Genes Involved in Salinity Stress in Wild Eggplant (Solanum torvum Swartz)

Gang Chen,Hua Wang,Jun-Yi Gai,Yue-Lin Zhu,Li-Fei Yang,Qian-Qian Liu,Gong-Chen Zhang,Guo-Hu Chen 한국원예학회 2012 Horticulture, Environment, and Biotechnology Vol.53 No.2

The objectives of this paper were to construct a full-length cDNA library and to isolate genes that confer salt tolerance from the leaves of salinity-tolerant wild eggplant variety, ‘Torvum Vigor’ (Solanum torvum Swartz). A full-length cDNA library from the leaves was successfully constructed by a switching mechanism at 5’-end of RNA transcript (SMART) approach and a long-distance PCR (LD-PCR) technique. The titer of the primary cDNA library was 3.6 ×106 cfu・mL-1 and that of the amplified library was 1.2 × 1010 cfu・mL-1. Gel electrophoresis results showed that most of the cDNA inserts ranged from 0.40 to 2.5 kb, with a recombination rate of 99%. A total of 427 randomly selected positive clones were sequenced. After removing the unsuccessful reads, 364 datasets were obtained and have been submitted to the NCBI Nucleotide Sequence Database under GenBank accession numbers JK265131-JK265494. Among the 364 submitted sequences, 74.45% of them contained full-length coding regions. BLASTX analysis revealed that 62.36% of the ‘Torvum Vigor’ expressed sequence tags (ESTs) possessed homology to known or putative proteins of other organisms. Seven genes that might be responsible for the encoding of known proteins in other organisms were identified to confer salt tolerance. This evidence demonstrated that the cDNA library constructed was a full-length library of high quality. It could be a useful resource for further research in the cloning of stress-related genes, which could be utilized in the genetic improvement of vegetable crops using transgenic technology.

Cloning and functional analysis of two GmDeg genes in soybean [Glycine max (L.) Merr.]

Xing Kong,Jingyao Zhang,Deyue Yu,Jun-Yi Gai,Shouping Yang 한국식물학회 2017 Journal of Plant Biology Vol.60 No.1

Although light is the ultimate substrate in photosynthesis, strong light can also be harmful and lead to photoinhibition. The DEG proteases play important roles in the degradation of misfolded and damaged proteins. In this study, two photoinhibition-related genes from soybean [Glycine max (L.) Merr.], GmDeg1 and GmDeg2, were cloned. Bioinformatics analysis indicated that these two proteases both contain a PDZ domain and are serine proteases. The expression levels of GmDeg1 and GmDeg2 increased significantly after 12 h of photooxidation treatment, indicating that GmDeg1 and GmDeg2 might play protective roles under strong light conditions. In in vitro proteolytic degradation assays, recombinant GmDeg1 and GmDeg2 demonstrated biological activities at temperatures ranging from 20°C to 60°C and at pH 5.0 to 8.0. By contrast, the proteases showed no proteolytic effect in the presence of a serine protease inhibitor. Taken together, these results provided strong evidence that GmDeg1 and GmDeg2 are serine proteases that could degrade the model substrate in vitro, indicating that they might degrade damaged D1 protein and other mis-folded proteins in vivo. Furthermore, GmDeg1 and GmDeg2 were transformed into Arabidopsis thaliana to obtain transgenic plants. Leaves from the transgenic and wild-type plants were subjected to strong light conditions in vitro, and the PSII photochemical efficiency (Fv/Fm) was measured. The Fv/Fm of the transgenic plants was significantly higher than that of the wild-type plants at most time points. These results imply that GmDeg1 and GmDeg2 would have similar functions to Arabidopsis AtDeg1, thus accelerating the recovery of PSII photochemical efficiency.

Enhanced Salt Tolerance of Transgenic Vegetable Soybeans Resulting from Overexpression of a Novel Δ1-Pyrroline-5-carboxylate Synthetase Gene from Solanum torvum Swartz

Gong-Chen Zhang,Wen-Li Zhu,Jun-Yi Gai,Yue-Lin Zhu,Li-Fei Yang 한국원예학회 2015 Horticulture, Environment, and Biotechnology Vol.56 No.1

Vegetable soybeans [Glycine max (L.) Merrill] are susceptible to salt stress and, thus, soil salinity can severely affect their growth and productivity. To enhance the salt tolerance of vegetable soybeans, a novel Solanum torvum Swartz Δ1-pyrroline-5-carboxylate synthetase gene (StP5CS, GenBank accession number: JN606861) that encodes a critical regulatory enzyme in proline biosynthesis was transformed into the cultivar NY-1001 using an Agrobacteriummediated transformation method. PCR and Southern blot analyses indicated that two independent T0 fertile transgenic plants were generated. The transgenic plants transmitted the transgenes into their T1 progeny in a 3:1 ratio. The T2 and T3 homozygous transgenic lines (HTLs) were examined for salt tolerance in pot and hydroponic cultures, respectively. The StP5CS overexpression conferred salt tolerance in T2 and T3 HTLs. Under NaCl stress conditions, the leaf scorch scores of T2 and T3 HTLs were significantly lower than those of wild-type (WT) plants. The plant height, leaf area, relative chlorophyll content, and number of fresh pods of T2 and T3 HTLs were significantly higher than those of WT plants. Compared with WT plants, T2 and T3 HTLs had significantly higher levels of proline and significantly lower levels of membrane lipid peroxidation. These results indicate that StP5CS overexpression in HTLs results in enhanced salt tolerance associated with higher levels of proline accumulation under salinity stress and that StP5CS can be utilized to improve salinity tolerance in vegetable crop genetic engineering.

Highly Efficient Shoot Regeneration from Cotyledonary Nodes of Vegetable Soybean

Qian-Qian Liu,Gang Chen,Jun-Yi Gai,Yue-Lin Zhu,Li-Fei Yang,Guo-Ping Wei,Cong Wang 한국원예학회 2010 원예과학기술지 Vol.28 No.2

풋콩(Glycine max (L.) Merrill) 6개 품종의 자엽절 절편체로부터 thidiazuron(TDZ)과 NAA 농도를 달리한 배지에서 신초의 효율적인 재분화를 조사한 후, 가장 효과가 좋은 생장조절물질 조합으로 배지 종류, 접종방법 및 품종별 신초 재분화율을 조사하였다. 또한 절편체를 배지에 수직 또는 수평으로 치상하는 방법과 B5, 1/2 B5, MS, 1/2 MS, MSB(MS salts + B5 organics) 등의 5가지 기본 배지에 관하여 시험하였다. B5 배지에 1㎎?L<SUP>-1</SUP> TDZ, 0.05㎎?L<SUP>-1</SUP> NAA, 5㎎?L<SUP>-1</SUP> AgNO₃를 첨가한 처리에서 6개 품종의 신초 재분화율이 55.3-88.9%로 높았다. ‘L?ling No. 1’의 경우 수직으로 치상하는 것이 수평으로 치상하는 것보다 신초 재분화율이 37.5% 더 높았다. 따라서 풋콩은 TDZ를 처리한 B5 배지에 자엽절을 수직으로 치상할 때 신초 재분화율이 높았다. To establish a highly efficient system for shoot regeneration in vegetable soybean (Glycine max (L.) Merrill), explants were obtained from six genotypes and adventitious shoots were regenerated from cotyledonary nodes cultured on medium supplemented with different concentrations of N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (thidiazuron TDZ) and 1-naphthylacetic acid (NAA). The most effective combination of plant growth regulators was selected first and then the effects of medium types, inoculation methods and genotypic differences on shoot regeneration were studied. The explants were inserted either vertically or horizontally into the medium and five basal media, including B5, ½ B5, MS, ½ MS, and MSB (MS salts and B5 organics), were tested. The shoot regeneration frequency of the six genotypes ranged from 53.5% to 88.9% and three of them reached 88.9%, 87.5% and 83.3%, respectively, on B5 medium supplemented with 1 ㎎?L<SUP>-1</SUP> TDZ, 0.05 ㎎?L<SUP>-1</SUP> NAA, and 5 ㎎?L<SUP>-1</SUP> AgNO₃. The shoot regeneration frequency of explants cultured on B5 medium was significantly higher than that of the other four basal media. The vertically inserted explants were found to yield a higher shoot regeneration frequency than that of horizontally inserted ones; the maximum difference of regenerating percentages between the two methods was 37.5%. In summary, TDZ was an efficient plant growth regulator for shoot induction. B5 medium and vertically inserted explants promoted shoot regeneration. We believe this highly efficient shoot regeneration system will provide foundation for the further transgenic studies in vegetable soybean.

Overexpression of rice phosphate transporter gene OsPT2 enhances nitrogen fixation and ammonium assimilation in transgenic soybean under phosphorus deficiency

Wen-Li Zhu,Li-Fei Yang,Shouping Yang,Jun-Yi Gai,Yue-Lin Zhu 한국식물학회 2016 Journal of Plant Biology Vol.59 No.2

Phosphorus (P) deficiency is one of the major factors that limit legume nodulation and nitrogen (N) fixation, and thus legume productivity. In our previous study, we showed that three T2 transgenic soybean lines overexpressing rice phosphate transporter gene OsPT2 showed enhanced tolerance to low P stress. This study aimed to determine whether OsPT2 overexpression would increase N2 fixation and ammonium assimilation in three T3 homozygous transgenic lines (HTLs) under P deficiency in pot culture. Under low inorganic phosphate (Pi) conditions, the P accumulation, total N and total ureide concentrations were significantly higher in the T3 HTLs than in the wild type (WT) plants. Further, the T3 HTLs showed significantly better plant growth performance and nodule development than the WT plants under low-Pi conditions. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression levels of GmENOD40-1, GmENOD40-2 (two early nodulin genes), and GmLba (one leghemoglobin gene) were significantly increased in T3 HTLs under P deficiency at 24 and 32 d after inoculation (DAI). The increased transcript levels of GmGS1β1 and GmGS1β2 (two cytosolic glutamine synthetase genes) in the T3 HTLs were consistent with the increase in glutamine synthetase (GS, EC 6.3.1.2) activity at 32 DAI. Our results indicated that the overexpression of OsPT2 in T3 HTLs enhances N2 fixation and ammonium assimilation activity under low P stress.

Overexpression of StNHX1, a Novel Vacuolar Na+/H+ Antiporter Gene from Solanum torvum, Enhances Salt Tolerance in Transgenic Vegetable Soybean

Guo-Hu Chen,Wenyan Wang,Li-Fei Yang,Jun-Yi Gai,Yue-Lin Zhu 한국원예학회 2014 Horticulture, Environment, and Biotechnology Vol.55 No.3

Soil salinity is the primary limiting factor affecting the growth and production of vegetable soybean[Glycine max (L.) Merrill] worldwide. Plants can survive in salinity conditions through the mechanism of sodium ion(Na+) compartmentalization, during which the vacuolar Na+/H+ antiporter (NHX) genes play a critical role. In the presentstudy, a novel vacuolar NHX gene from Solanum torvum Swartz ‘Torvum Vigor’ (StNHX1; GenBank accessionnumber: JN606860.1) was isolated and transferred into the vegetable soybean ‘Xinliaoxian’ through an Agrobacteriummediatedprotocol. The PCR and southern blot analyses confirmed the successful integration of the exogenous genes. The GUS assay and semi-quantitative RT-PCR analysis showed that the alien genes were inherited by the progeniesand that the StNHX1 gene was overexpressed in the T3 generation. The overexpression of this gene conferred hightolerance to salt stress. Under 100 mM NaCl conditions, the scorch scores and the Na+ and malondialdehyde (MDA)contents of the leaves of the transgenic lines were significantly lower as compared with the wild-type (WT) plants,while the K+/Na+ ratio, the contents of K+, relative chlorophyll and relative water, and some morphological traits of thetransgenic plants were significantly higher than those of the WT. These results indicate that the overexpression ofStNHX1 enhances the salt tolerance of vegetable soybean and that StNHX1 is one of the promising target genes thatcan be manipulated to improve the salinity tolerance of crops.