Cloning and functional study of GmRPI2, which is the critical gene of photosynthesis in soybean.

Light provides energy for photosynthesis and is also an important environmental signal that regulates plant growth and development. Ribose-5-phosphate isomerase plays a crucial role in photosynthesis. However, ribose-5-phosphate isomerase has yet to be studied in soybean photosynthesis. To understand the biological function of GmRPI2, in this study, GmRPI2 was cloned, plant overexpression vectors and gene editing vectors were successfully constructed, and transformed into recipient soybean JN74 using the Agrobacterium-mediated method. Using qRT-PCR, we analyzed that GmRPI2 gene expression was highest in leaves, second highest in roots, and lowest in stems. Promoter analysis revealed the presence of multiple cis-acting elements related to light response in the promoter region of GmRPI2. Compared with the control soybean plants, the net photosynthetic rate and transpiration rate of the overexpression lines were higher than those of the control and gene editing lines, while the intercellular CO2 concentration was significantly lower than that of the control and gene editing lines; the total chlorophyll, chlorophyll a, chlorophyll b contents and soluble sugar contents of the overexpression plants were significantly higher than those of the recipient and editing plants, indicating that the GmRPI2 gene can increase The GmRPI2 gene can increase the photosynthetic capacity of soybean plants, providing a theoretical basis and genetic resources for improving soybean yield by regulating photosynthetic efficiency.

Enhancement of root sulfur metabolic pathway by overexpression of OAS-TL3 to increase total soybean seed protein content.

Sulfur is essential for plant growth, and the uptake of sulfate by plant roots is the primary source of plant sulfur. Previous studies have shown that the OAS-TL gene is a key enzyme in the sulfur metabolic pathway and regulates cysteine (Cys) synthase production. However, the interaction mechanism of the glycine max OAS-TL3 Cys synthase (OAS-TL3) gene on soybean root morphology construction and seed protein accumulation is unclear. This study shows that mutant M18 has better root growth and development, higher seed protein content, and higher methionine (Met) content in sulfur-containing amino acids than wild-type JN18. By transcriptome sequencing, the differentially expressed OAS-TL3 gene was targeted in the mutant M18 root line. The relative expression of the OAS-TL3 gene in roots, stems, and leaves during the seedling, flowering, and bulking stages of the OAS-TL3 gene overexpression lines is higher than that of the recipient material. Compared to the recipient material JN74, the enzymatic activities, Cys, and GSH contents of OAS-TL are higher in the sulfur metabolic pathway of seedling roots. The receptor material JN74 is exogenously applied with different concentrations of reduced glutathione. The results demonstrate a positive correlation between reduced glutathione on total root length, projected area, surface area, root volume, total root tip number, total bifurcation number, and total crossing number. The Met and total protein contents of sulfur-containing amino acids in soybean seeds of the OAS-TL3 gene overexpression lines are higher than those of the recipient material JN74, while the gene-edited lines show the opposite results. In conclusion, the OAS-TL3 gene positively regulates soybean root growth, root activity, and the content of Met in the seeds through the OAS-TL-Cys-GSH pathway. It breaks the limitation of other amino acids and facilitates the increase of total seed protein content. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01348-y.

The GmXTH1 gene improves drought stress resistance of soybean seedlings.

In order to study the role of GmXTH1 gene in alleviating drought stress, soybean seeds with GmXTH1 gene were transferred by T4 treated with PEG6000 concentration of 0%, 5%, 10%, and 15% respectively. The germination potential, germination rate, germination index, and other indicators were measured. The results showed that the germination potential, germination rate, and germination index of OEA1 and OEA2 strains overexpressed in T4 generation were significantly higher than those of the control material M18. After 0-day, 7-day, and 15-day drought stress, the analysis of seedling phenotypes and root-shoot of different T4 generation transgenic soybean lines showed that under stress conditions, the growth of GmXTH1 overexpression material was generally better than that of the control material M18. The growth of GmXTH1 interference expression material was generally worse than that of the control material M18, with significant differences in plant phenotypes. The root system of GmXTH1 overexpressed material was significantly developed compared with that of the control material M18. The analysis of physiological and biochemical indexes showed that the relative water content and the activity of antioxidant enzymes (superoxide dismutase and peroxidase) of GmXTH1 transgenic soybean material were significantly higher than those of the control material M18, and the accumulation of malondialdehyde was lower under the same stress conditions at seedling stage. Fluorescence quantitative PCR assay showed that the relative expression of GmXTH1 gene in transgenic soybean was significantly increased after drought stress. The results showed that the overexpression of GmXTH1 could increase the total root length, surface area, total projection area, root volume, average diameter, total cross number, and total root tip number, thereby increasing the water intake and reducing the transpiration of water content in leaves, thus reducing the accumulation of MDA and producing more protective enzymes in a more effective and prompt way, reducing cell membrane damage to improve drought resistance of soybean.

Soybean plants expressing the Bacillus thuringiensis cry8-like gene show resistance to Holotrichia parallela.

BACKGROUND: Cry8-like from Bacillus thuringiensis (Bt) encodes an insecticidal crystal (Cry) protein. Holotrichia parallela (Coleoptera: Scarabaeoidae), commonly known as the dark black chafer, is a troublesome pest of soybean (Glycine max). To test whether cry8-like can confer resistance against H. parallela to soybean, we introduced cry8-like from the Bt strain HBF-18 into soybean cultivar Jinong 28. RESULTS: Quantitative reverse transcription-PCR analysis demonstrated that cry8-like was expressed most highly in soybean leaves. In addition, Southern blot assays revealed that one copy of the integrated fragment was present in the transformed plants. Eight independent cry8-like transgenic lines were subsequently fed on by H. parallela. Under H. parallela feeding stress, the survival rates of the non-transgenic plants were 92% lower than those of the transgenic plants. The mortality rate of H. parallela increased when the larvae fed on the roots of T1 transgenic soybean plants. Moreover, the surviving larvae were deformed, and their growth was inhibited. CONCLUSIONS: Collectively, our data suggest that transgenic soybean plants expressing the cry8-like gene are more resistant to H. parallela than non-transgenic plants and that transgenic expression of the cry8-like gene may represent a promising strategy for engineering pest tolerance. The events generated in this study could thus be utilized in soybean breeding programs.

Construction of a chalcone reductase expression vector and transformation of soybean plants.

The present study aimed to clone the soybean chalcone reductase 3 (CHR3) and create a recombinant expression vector pCAMBIA3300­CHR3 containing Bar resistance gene as a selection marker, and then obtain transgenic soybean plants using Agrobacterium infection. The plant expression vector pCAMBIA3300­CHR3 was transferred into soybean receptor plants, Jinong 17 and Jilin 30. Polymerase chain reaction (PCR) and Southern blotting were used to confirm the positive transgenic plants. Additionally, reverse transcription­quantitative PCR (RT­qPCR) was used to detect CHR3 expression and isoliquiritigenin content was measured using high­performance liquid chromatography (HPLC) in the transgenic offspring. Soybean CHR3 (932 bp fragment) was successfully cloned into the plant expression vector pCAMBIA3300­CHR3, which was subsequently transferred into soybean receptor plants. In the T1 generation positive plants were validated by PCR analysis, including eight Jinong 17 and five Jilin 30 transgenic plants; Southern blotting demonstrated that the functional components of the pCAMBIA3300­CHR3 vector had been integrated into the soybean genome; RT­qPCR results demonstrated that the expression of CHR3 mRNA was increased by 2 to 20­fold in the transgenic plants compared with the non­transgenic soybean plants. Furthermore, the isoliquiritigenin content was increased by 8.56% in the transgenic Jinong 17, compared with control plants, as detected by HPLC. The CHR3 gene can produce isoliquiritigenin, a precursor of daidzein, which in turn can improve the ability of soybean to resist phytophthora root rot.

Upscale production of a recombinant cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli.

Cyclodextrin glucanotransferase (CGTase) is an important enzyme with multiple functions in starch utilization. In the present study, a fermentation process for the production of CGTase from Escherichia coli harboring the recombinant plasmid pET28b(+)-CGTase was investigated and optimized. The optimal fermentation and expression conditions were 10.0 g/L glycerol, 20.0 g/L tryptone, and 10.0 g/L yeast extract with an initial pH of 7.0, an IPTG concentration of 0.1 mM and an induction temperature of 28 °C for 10 h. The resulting CGTase activity reached up to 36.4 U/L and was 2.1-fold higher than before optimization. Under these optimal fermentation conditions, the up-scaled fermentation was carried out in a 500-L fermentor, and a CGTase activity of 45.2 U/L was achieved. This study provides a foundation for the industrial production of CGTase.

Structure of C-terminal Domain of Peptidyl-prolyl cis-trans Isomerase from Pseudomonas syringae pv. Tomato str. DC3000 at 1.6Å Resolution.

BACKGROUND: Peptidyl-prolyl cis-trans isomerase (PPIase) accelerates the intrinsically slow conversion between cis- and trans- configurations of proline, thus affecting backbone conformation and altering the direction of peptide chains. PPIase from Pseudomonas syringae pv. Tomato (PSPTO) DC3000 (PSPTO-PPIase) is considered to belong to the FKBP subfamily of PPIase. OBJECTIVE: To solve the high resolution structure of the PSPTO-PPIase, and to explore its potential function in plants pathogen PSPTO DC3000. METHOD: The PSPTO-PPIase was expressed in E.coli and purified through ion exchange and size exclusion chromatography. While only the C-terminal domain of PSPTO-PPIase was successfully crystalized, and its structure was solved to 1.6 Å resolution by molecular replacement method. RESULTS: Structural comparison showed that PSPTO-PPIase adopts a similar overall fold with microphage infectivity potentiators (MIPs), which also belong to the FKBP subfamily of PPIase. In addition, the BIAcore result confirmed that PSPTO-PPIase can bind an immunosuppressive drug FK506 as some other FKBP subfamily members do. CONCLUSION: Our results suggested that PSPTO-PPIase may function in a similar manner to virulent factor MIPs during pathogenesis. And the immunosuppressive drugs FK506 and rapamycin binding to PSPTO-PPIase potentially interferes and inhibits the plant pathogen PSPTO DC3000. In addition, the amino acids with short side chains in the fourth loop (L4) of PSPTO-PPIase may account for its variable roles in the respective pathogen.

[Cloning and functional analysis of SCTF-1 encoding a C2H2-type Zinc finger protein from soybean].

The zinc finger protein is one of the proteins with finger-like domain. Some of them are transcription factors which play important role in plant growth and plant resistance to abiotic stresses. In this paper, a novel C2H2-type zinc finger protein gene SCTF-1 (GenBank accession number JQ692081) was isolated from soybean (Glycine max (L.) Merr.) This gene has a 699 bp ORF (open reading frame) with no intron and encodes a 24.9 kDa protein with 233 amino acids. Its isoelectric point (pI) is 8.33. The SCTF-1 protein contains two typical C2H2-type zinc finger domains. Both of them have highly conserved amino acid sequence-QALGGH which is a particular characteristic of plant. Transient expression of the GFP-SCTF-1 protein in onion epidermal cell showed that SCTF-1 was localized in cell nuclei. RT-PCR results showed that SCTF-1 gene was expressed with high levels in flowers and leaves in soybean, but low in roots and stems. The expression of SCTF-1 gene was strongly induced by low temperature in the soybean seedlings. Overexpression of SCTF-1 enhanced cold tolerance of transgenic tobacco (Nicotiana tabacum L.) compared to the control.

Identification of a novel mutation in FAD2B from a peanut EMS mutant with elevated oleate content.

A Virginia type peanut mutant with more than 60% oleate content (E2-4-83-12) was selected from an EMS mutagenized population of LF2 (an export type peanut cultivar with 44.2% oleate) by near infrared reflectance spectroscopy. Cloning and sequencing of FAD2B from LF2 and E2-4-83-12 identified a novel mutation (C313T in the coding region) causing an H105Y substitution in the first histidine box of the FAD2B protein. GC-MS analysis of fatty acids in yeast cells harboring pYES2 with the mutated FAD2B detected no linoleate, confirming that FAD2B from E2-4-83-12 was dysfunctional. Loss-of-function FAD2A and FAD2B together contributed to elevated oleate phenotype of the peanut EMS mutant.

[Reducing the maize amylopectin content through RNA interference manipulation].

To regulate the biosynthesis of maize starch, gene segment of starch branch enzyme (SBE) in maize was cloned and an expression vector of small interference RNA with the fragment of antisense gene + sense gene (pCJSBE2b, Fig.1) was constructed. The expression vector of pCJSBE2b was transformed into maize inbred lines by using pollen-tube pathway. Results of PCR amplification and Southern blotting indicated that the aimed gene with the fragment of antisense gene + sense gene had integrated into receptor maize genome (Fig.4,5) and inherited steadily in the transgenic plants (Table 1). Northern hybridization analysis showed that the aimed gene transcribed normally in the transgenic plants, which resulted in a reduction of endogenous SBE mRNA (Fig.6). The analysis of the activity of starch branching enzyme showed that the enzyme activity decreased obviously in the transgenic plants and maximum decrease was 85% compared to the control plants (Fig.7a). Although the total starch content of transgenic plants showed no change compared to the control plants but the amylose content increased to 50% (Fig.7b) in transgenic plants about 27% higher than receptor maize plants.