Identification Of MhSCL29 Gene,a Member Of The GRAS Family In Malus Hupenensis And Its Response To Nitrate

Nitrogen fertilizers have made an important contribution to apple production,but imprope r application has also brought about problems such as decreased utilization,increased costs,a nd environmental pollution.Nitrate is an important member of nitrogen fertilizers.Malus hup enensis is an excellent apple rootstock.Cultivation of apples needs to respond to nitrate throu gh its rootstock.GRAS is a plant-specific transcription factor,and SCL29 is a member of the SHR subfamily of the GRAS family.Based on the analysis of the differential response of Ma lus hupenensis to different concentrations of nitrate,this study conducted a genome-wide anal ysis of the GRAS family in apples.MhSCL29 gene was used to overexpress MhSCL29 in call us and transiently expressed in roots to analyze the physiological and biochemical changes un der nitrate treatment,and to explore the role of MhSCL29 gene in Malus hupenensis in respo nse to nitrate.The main results are as follows:1.The biomass,chlorophyll and carotenoid contents,total length of taproot and root,root volume,root tip number and root activity of seedlings of Malus hupenensis were significantl y different under 1/2 hoagland nutrient solution containing 0 m M,0.15 m M,3 m M and 15 m M nitrate.With the increase of nitrate concentration,the activities of nitrate reductase and glut amine synthase in roots were gradually increased,and the expressions of NRT1.1,NRT2.1 and D27 were gradually down-regulated.2.78 GRAS transcription factor sequences with GRAS characteristic domains were identi fied from the apple genome（GDDH13）,there are 9 pairs of tandem duplicating genes and 38 pairs of segmental duplicating genes,and all 78 Md GRASs genes contain light-responsive ele ments,among which,Md GRAS40（Md SCL29）contains elements related to auxin response,ci rcadian rhythm control,defense and stress response,low temperature response,methyl jasmo nate response,wound response,and regulation of zein metabolism.3.Nine SHR subgroup genes in GRAS family were identified from Malus Hupenensi.Exc ept MhGRAS20 and MhGRAS40（MhSCL29）,transcription levels of most SHRs were higher in shoots and lower in roots,but all SHR subgroup genes in roots responded to different concent rations of nitrate.The expression of MhGRAS40（MhSCL29）decreased with the increase of ni trate concentration.4.The length of the open reading frame of the MhSCL29 gene in Malus hupenensis is 1656 bp,encoding 553 amino acids.Using the technique of transient transformation of tobacco le aves,it was found that the gene MhSCL29 is located in the nucleus.5.Under the treatment of strigolactone analog GR24,the change trend of MhSCL29 gene in Malus hupenensis roots was similar to that of D27,and the expressions of nitrogen absorpt ion and assimilation related genes MhNRT1.1,MhNRT2.1,MhNR2,MhNIR,MhGS2,MhGO GAT Overall increase.6.Apple callus was treated on callus subculture medium containing 0 M,0.02 M,0.04 M and 0.08 M nitrate for 15 d,the color of callus was darker under 0.08 M nitrate treatment,and cell viability was significantly reduced;The fresh weight of apple callus expressing MhSCL29 was significantly lower than that of the wild type,but the cell viability was significantly high er than that of the wild type.7.Overexpression of MhSCL29 increased the content of nitrate nitrogen,nitrite nitrogen a nd a Monium nitrogen and the activities of NR,NIR and GOGAT in apple callus under differe nt nitrate treatments,promoted the expression of NR2,NIR,GS2 and GOGAT genes,and inter fered with The expression of NRT1.1 and NRT2.1 promotes the expression of the key gene D27 for strigolactone synthesis,inhibits the expression of GS1.1 and GS1.2,and reduces the acti vity of glutamine synthase.8.Transient expression of MhSCL29 increased nitrate nitrogen,nitrite nitrogen and a Moni um nitrogen contents and NR,NIR and GOGAT activities in Malus hupenensis roots under d ifferent nitrate treatments,inhibited GS activity,and promoted Malus hupenensis roots D27,NRT1.1,The expression of NRT2.1,NR2,NIR,GS2 and GOGAT was suppressed,and the exp ression of GS1.1 and GS1.2 was inhibited.