Expression Of Agronomic Traits In The Offspring Of SoSUT5 Transgenic Sugarcane

Sugarcane is a C₄ high photosynthetic capacity sugar crop with complex genetic background.The gene fragment size and integration mode are important factors affecting the genetic stability of transgenic offspring and associated to stable inheritance of target traits in the crop.The sucrose transporter gene（SUT）is closely related to sucrose metabolism,and its encoded protein has sucrose transport activity.It is responsible for the transmembrane transport of sucrose,and plays a key role in the phloem-mediated source transport and the supply of sucrose to the sink organs.The purpose of this study is to observe the growth,agronomic traits,juice rate,juice gravity purity,juice sucrose content,sucrose%cane and other traits in the So SUT5 transgenic generations T2（plant cane）,T2R1（1st ratoon of T2）,T2M22（22-month-old T2 plant cane）and T3（plant cane）after transformed into sugarcane variety B8,to perform transcriptome sequencing with the internodes of sugarcane stems at processing mature stage of the T3 sugarcane generations,and to verify the results of the transcriptome data and analyze the function of So SUT5 gene in sugar accumulation,and to provide a reference for further research on the genetically modified sugarcane.The main results were as follows.1.Through conventional PCR detection of transgenic sugarcane leaf+1,the presence of the marker,bar gene,in the transgene carrier was also detected in the transgenic plants of T2 and T3.The test results indirectly indicated that the So SUT5 gene transformed into sugarcane variety B8 had not been lost in the offspring.After screening the herbicide resistance,the color of WT leaves was getting yellow,but that of each transgenic sugarcane lines did not change under the same conditions.The phenotypic observations confirmed that the transformed So SUT5 gene still existed in the transgenic sugarcane.2.Compared with the WT,the T2 and T3 grown under natural environmental conditions significantly changed the stalk diameter and field Brix in the So SUT5 transgenic sugarcane（lines L1,L2,L3,and L6）.The emergence rate in the four transgenic sugarcane lines was lower,the stalk diameter was significantly smaller,and the smut incidence rate was higher as compared with WT.Except for L6,the tillering rate and millable stalks in the So SUT5transgenic sugarcane lines were higher than those in WT.At processing mature stage,the stalk weight in the So SUT5 transgenic lines was significantly lower than that in WT.The sugarcane sucrose%cane and juice gravity purity were reduced in varying degrees,while the juice rate enhanced as compared with WT.The sucrose%cane in the So SUT5 transgenic lines was lower than that in WT at T2,T2R1,T2M22 and T3.These results suggested that the overexpression of So SUT5 gene under natural environmental conditions may negatively regulate the growth and sugar content,and increase the smut incidence rate in sugarcane.3.The sucrose phosphate synthase（SPS）activity and sucrose synthase in synthesis direction（SSs）activity in leaf+1 of 4 transgenic sugarcane lines planted under natural conditions were significantly lower than that in WT at processing mature stage.Sugarcane was obviously affected by the transfermed So SUT5 gene,and its natural resistance became worse under natural conditions..The effect of overexpression of So SUT5 gene on SSs enzyme activity in the transgenic sugarcane was significant compared with WT.The acid invertase（AINV）activity in leaf+1 of the 4 transgenic sugarcane lines was significantly higher than WT at processing maturity stage,and it was also higher than that at seedling stage.In addition to L6,the neutral invertase（NINV）activity in leaf+1of the other transgenic sugarcane lines was higher than that in WT at processing mature stage.4.High-throughput transcriptome sequencing was performed with cane internodes at processing mature stage,the difference in gene expression between transgenic sugarcane and WT was analyzed.A total of 270273 transcripts and117576 unigenes were obtained by sequencing,and the reliability of transcriptome sequencing results was verified by q RT-PCR.It was found that L1and WT had 872 differentially expressed genes,of which 703 were up-regulated and 169 were down-regulated;L2 and WT had 720 differentially expressed genes,of which 529 were up-regulated and 191 were down-regulated.Compared with WT,L1 and L2 had 487 common differentially expressed genes.The cluster analysis of differential gene expression level further showed that there was a big difference between So SUT5 transgenic sugarcane and WT.The overexpression of So SUT5 gene has a significant impact on the gene expressions in sugarcane.GO enrichment analysis revealed that the differential genes were mainly enriched in the modification by symbiont of host morphology or physiology,modification of morphology or physiology of other organism involved in symbiotic interaction,secondary metabolite biosynthetic process,cellulose biosynthesis process,cellulose metabolism process,biological functions such as cytoskeletal organization and interactions between species.KEGG pathway enrichment analysis was performed on the differentially expressed genes,and it was found that the genes were mainly enriched in the pathway of DNA mismatch repair,related pathways of life activities such as stress response,signal transduction,etc.In conclusion,the transgenic sugarcane had smaller stems,reduced sugar content,increased incidence of smut,and reduced disease resistance.Based on the results of the present study,So SUT5 gene may negatively regulate sugarcane growth and sugar accumulation,and increase the incidence of smut,which suggested that knocking out of this gene may improve sugarcane growth,yield and quality traits.