Optimization of Agrobacterium tumefaciens-Mediated Genetic Transformation of Maize

Immature embryos of inbred maize(Zea mays)lines(H8183,H8184,and H8185)were used for Agrobacterium infection.We used theβ-glucuronidase gene(GUS)as the target gene and the glufosinate resistance gene(bar)as the selection marker.We conducted research on several aspects,such as different genotypes,coculture conditions,screening agent concentrations,and concentrations of indole-3-butytric acid(IBA),6-benzylaminopurine(6-BA),and ascorbic acid(Vc)in the differentiation medium.We optimized the genetic transformation system,and the obtained results indicated that among the three lines studied,the induction rate of H8185 was the highest at 93.2%,followed by H8184,with H8183 having the lowest induction rate(80.1%).The best coculture method was that using the N6 coculture medium layered with a sterile filter paper.Using orthogonal analysis,we found that the optimal combination of the three factors in the differentiation medium was A_(3)(1 mg mL^(−1)IBA),B_(3)C_(1)(1.6 mg mL^(−1)6-BA),and D_(3)(1.5 mg mL^(−1)Vc).Through GUS staining analysis,Bar test-strip analysis,and polymerase chain reaction,five transgenic plants were finally obtained.This study established the optimal conditions for genetic transformation in maize.

Meta-Analysis of Flowering-Related Traits and Mining of Candidate Genes in Maize

Maize flowering is an important agronomic character,which is controlled by quantitative trait loci(QTL).Over the years,a large number of flowering-related QTL have been found in maize and exist in public databases.However,combining these data,re-analyzing and mining candidate loci and fine mapping of flowering-related traits to reduce confidence intervals has become a hot issue in maize.In this study,the QTL of 6 important agronomic traits of maize flowering were collected from 15 published articles,including flowering period(DA),Days to tasseling(DTT),Days to silking(DS),Days to pollen shedding(DTP),anthesis-silking interval(ASI)and the photosensitive(PS).Through meta-analysis,622 QTL were integrated into 26 meta-QTLs(MQTL).Finally,the candidate genes related to maize flowering(Gene IDs:ZM00001D005791,ZM00001D019045,ZM00001D050697,ZM00001D011139)were identified by Gene Ontology(GO)enrichment and hierarchical cluster analysis of expression profile.Based on the results of this study,the genetic characteristics of maize flowering traits will be further analyzed,which is of great significance to guide the improvement of important agronomic characters and improve the efficiency of breeding.

METTL3 regulates glucose transporter expression in placenta exposed to hyperglycemia through the mTOR signaling pathway

Background:Alterations in the placental expression of glucose transporters(GLUTs),the crucial maternal-fetal nutrient transporters,have been found in women with hyperglycemia in pregnancy(HIP).However,there is still uncertainty about the underlying effect of the high-glucose environment on placental GLUTs expression in HIP.Methods:We quantitatively evaluated the activity of mammalian target of rapamycin(mTOR)and expression of GLUTs(GLUT1,GLUT3,and GLUT4)in the placenta of women with normal pregnancies(CTRL,n=12)and pregnant women complicated with poorly controlled type 2 diabetes mellitus(T2DM,n=12)by immunohistochemistry.In addition,BeWo cells were treated with different glucose concentrations to verify the regulation of hyperglycemia.Then,changes in the expression of GLUTs following the activation or suppression of the mTOR pathway were also assessed using MHY1485/rapamycin(RAPA)treatment or small interfering RNA(siRNA)-mediated silencing approaches.Moreover,we further explored the alteration and potential upstream regulatory role of methyltransferase-like 3(METTL3)when exposed to hyperglycemia.Results:mTOR,phosphorylated mTOR(p-mTOR),and GLUT1 protein levels were upregulated in the placenta of women with T2DM compared with those CTRL.In BeWo cells,mTOR activity increased with increasing glucose concentration,and the expression of GLUT1,GLUT3,and GLUT4 as well as GLUT1 cell membrane translocation were upregulated by hyperglycemia to varying degrees.Both the drug-mediated and genetic depletion of mTOR signaling in BeWo cells suppressed GLUTs expression,whereas MHY1485-induced mTOR activation upregulated GLUTs expression.Additionally,high glucose levels upregulated METTL3 expression and nuclear translocation,and decreasing METTL3 levels suppressed GLUTs expression and mTOR activity and vice versa.Furthermore,in METTL3 knockdown BeWo cells,the inhibitory effect on GLUTs expression was eliminated by activating the mTOR signaling pathway using MHY1485.Conclusion:High-glucose environment-induced upregulation of METTL3 in trophoblasts regulates the expression of GLUTs through mTOR signaling,contributing to disordered nutrient transport in women with HIP.