Effects Of Overexpression And Inhibition Of Expression Of AS Gene On Growth And Physiology Of Poplar

Asparagine synthetase(AS)is one of the key enzymes in the primary nitrogen assimilation process.Its product asparagine is the main carrier of plant nitrogen transport.In this study,two transgenic plants with overexpressing PnAS1(Populus alba×Populus glandulosa)were used to screen two test materials with high expression in aboveground and root tissues by qRT-PCR.Under different nitrogen supply levels,the growth indexes and physiological and biochemical indexes related to nitrogen metabolism of PnASl 84K poplar plants and wild-type WT were determined.An RNAi plant inhibitory expression vector of the AS]gene was constructed and an 84K Yang transgenic line which inhibited the expression of the AS1 gene was obtained.In addition,by searching the poplar genome,nine aspartate aminotransferases(the catalytic product aspartate,the only receptor for asparagine synthesis)family genes closely related to aspartate metabolism were identified and A brief analysis of bioinformatics is carried out.The main findings are as follows:(1)Nine aspartate aminotransferase family genes(PtASPAT)were identified from the poplar genome,including 7 eukaryotic ASPAT genes(PtASPAT1?PtASPAT7)and two prokaryotic ASPAT genes(PtASPAT8-PtASPAT9).The expression levels of 9 PtASPAT family genes were detected by real-time fluorescent quantitative PCR.The results showed that the expression of PtASPAT in roots was higher than that in other parts(leaves and stems);the expression of PtASPAT1 in roots and leaves was basically the same;the expression of PtASPAT4 and PtASPAT8 in roots was significant,and it was speculated that PtASPAT4 might be primary nitrogen in poplar roots.The assimilation process plays an important role.(2)The obtained high-transgenic transgenic lines(OEAS1-11 and OEAS 1-12 strains)were treated with three nitrogen levels(NH4NO3 concentrations of 0.05 mmol,1 mmol and 5 mmol,respectively)to determine growth indicator strains.High,ground diameter and leaf area;physiological and biochemical indicators related to nitrogen metabolism,GOT activity,soluble protein content and soluble sugar content,and total nitrogen content of plant total nitrogen.The results showed that the leaves of over-expressed plants under low nitrogen conditions(0.05 mmol NH4NO3)were darker and greener and the total number of leaves increased,especially OEAS1-12.The leaf area of OEAS1-11 and OEAS1-12 was higher than WT under the treatment conditions of 1 mmol and 5 mmol NH4NO3.However,the plant height and ground diameter of OEAS1-11 and OEAS1-12 were not significantly different from those of wild type WT under the three nitrogen treatment conditions.The results of physiological parameters related to nitrogen metabolism showed that under the treatment of 0.05 mmol NH4NO3,the OEAS1-11 strain had higher N content and lower C/N ratio than the wild type WT,but the total C content was not significantly different.Unlike the OEAS1-11 strain,the OEAS1-12 strain exhibited a higher C/N ratio.The GOT activity of OEAS1-11 and OEAS1-12 strains was higher than that of WT,in which the GOT activity in the aerial part was between 4 and 5 times that of WT,and between 2 and 3 in the root,the difference was significant;OEAS1-11 The content of soluble sugar in the aboveground part was significantly lower than that in WT,but it was significantly higher than that in WT.The soluble sugar content of OEAS1-12 was not significantly different from that of WT.The soluble protein of aboveground parts of OEAS1-11 and OEAS1-12 was significantly higher than that of wild.Type WT;Under the conditions of 1 mmol NH4NO3 and 5 mmol NH4NO3,the soluble sugar content of OEAS1-11 and OEAS1-12 in roots was higher than that of WT in OEAS1-11 and OEAS1-12,and the difference was significant.Under the condition of 1 mmol NH4NO3 treatment,the aboveground GOT activity was higher.The OEAS1-12 strain was significantly higher than WT;the GOT enzyme activity OEAS1-11 strain was significantly higher than WT;the aboveground soluble protein content OEAS1-11 was significantly higher than WT;the soluble protein content in roots OEAS1-11,OEAS1-12 was significantly higher than WT;over-expressing PnAS1 gene plants changed the balance of CN in their own plants,and 0.05 mmol NH4NO3 treatment was the most obvious.The experimental results need further study.(3)Expression of genes related to asparagine metabolism:semi-quantitative detection of ASPAT,AsnAT,ASPG,GS,GOG AT related genes for asparagine synthesis showed that over-expressed plants transgenic with AS gene enhanced ASPAT4,ASPAT8,ASPG1 ASPG2,ASPG4 and GS2 gene expression levels.Low nitrogen level aspartate aminotransferase GOT activity may be due to increased expression of ASPAT4 and ASPAT8;in addition,elevated GS2 indicates potential for glutamine synthesis;glutamine as asparagine synthesis The amide donor,increased glutamine,promotes asparagine synthesis.The asparaginase genes ASPG1,ASPG2,and ASPG4 are elevated,which may promote the hydrolysis of asparagine to aspartic acid.(4)The AS1 gene RNAi expression vector was constructed.After transforming 84K poplar,15 resistant strains of AS1 gene were obtained,and 6 of them were detected by PCR and real-time PCR.It was determined that the foreign DNA had been integrated into the 84K poplar genome.The expression levels of AS1 gene in five transformants were decreased to some extent,and the inhibitory effects of IEAS1-2 and IEAS1-3 plants were the most significant.The results of determination of physiological indicators related to nitrogen metabolism indicate that:The content of soluble protein of IEAS1-2 and IEAS1-3 was lower than that of wild type WT under the treatment of 0.05 mmol NH4NO3,among which IEAS1-3 was significantly different;the soluble sugar content of aboveground part of IEAS1-2 and IEAS1-3 was lower than WT.Among them,IEAS1-2 was significantly lower than WT;the GOT activity in roots was significantly lower than that of IEAS1-2 and IEAS1-3,and there was no significant difference in GOT activity in aboveground part;1 mmol NH4NO3 and 5 mmol NH4NO3 treatment,aboveground part There was no significant difference in soluble protein content between IEAS1-2 and IEAS1-3 compared with WT.The soluble protein contents of roots IEAS1-2 and IEAS1-3 were significantly lower than WT.Under the condition of lmmol NH4N03 treatment,the soluble sugars IEAS1-2 and IEAS1-3 in the aerial part and root were lower than WT,the difference was not significant;the GOT activity was lower than that of WT,the difference was significant;under the condition of 5mmol NH4NO3 treatment,the soluble in root The sugars IEAS1-2 and IEAS1-3 were lower than WT,and there was no significant difference in the above-ground soluble sugar.Asparagine synthesis related genes ASPAT,AsnAT,ASPG,GS,GOGAT semi-quantitative detection inhibited expression of transgenic plants IEAS1-2 and IEAS1-3 enhanced ASPAT1 expression,ASPG4 expression level decreased,ASPG1 and ASPG2 expression levels The decrease is presumed to inhibit the hydrolysis of asparagine to aspartic acid.