The Research About The Mechanism Of Transgenic Rice Plant With Overexpressing C₄ PEPC From Maize Under Low Nitrogen

Nitrogen is a constituent of proteins,nucleic acids,phospholipids and other organic compounds,which is an important plant nutrient in the vegetative and reproductive growth of plants.At the same time,nitrogen is the content of chlorophyll,chloroplast development,light synthase activity,so nitrogen plays an important role in photosynthesis and crop yields.Rice is one of the most important food crops,provides energy for nearly half the world's population.Nearly a decade,rice breeding increased yield through traditional means such as the increase of nitrogen fertilizer applied,but which is still limited.As we all know,photosynthesis and yield are closely related.Compared with C3 plants,C4 plants have a rosette structure and PEPC enzyme,CO2 can be concentrated in the same environment.So many scholars introduced C4 model into C3-tyPe crop,which greatly improved the traits such as photosynthesis and yields.Ku transferred phosphoenlpyruvate carboxylase(PEPC;EC4.1.1.31)gene from maize with Agrobacterium mediated to the Kitaake of japonica rice varieties,successful high expression,and performance improvement of photosynthetic characteristics and grain yield.Moreover,PC can be tolerant to low nitrogen condition,but we know litter about the mechanism.This paper studies the effect of long-term low nitrogen in pot on production,photosynthesis,physiological and enzymes activity of transgenic rice(PC)overexpressing the maize C4-PEPC gene and wild-type(WT)in succession two years;the effect of short-term low nitrogen in hydroponic on enzymes activity of carbon and nitrogen metabolism,photorespiration and expression of related genes of transgenic rice(PC)overexpressing the maize C4-PEPC gene and wild-type(WT),to revel the mechanism of PC response to low nitrogen level,The main results are as follows:1.In the 300 kg/hm2(nitrogen,MN)conditions,PC relative to WT rice yield is increased,but no significant difference at 65 kg/hm2(low nitrogen,LN))treatment conditions.Tiller number,leaf weight,stem weight of PC were significantly improved under low nitrogen condition compared with WT.This shows that,relative to the WT,PC under low nitrogen condition improve the photosynthetic product to maintain astable biomass.Furthermore,the leaf of PC accumulated more soluble sugar in lower nitrogen conditions which may be related to photosynthesis increased.2.To reveal transgenic rice with high expression of maize C4.PEPC photosynthetic performance under long-term low nitrogen condition,we measure photosynthesis characteristics,SPAD value,PEPC,Rubisco,NR and GS activities of flag leaves at different flowering stage under different nitrogen applications(normal nitrogen 300 kg/hm2,low nitrogen 65 kg/hm2).The results showed that the net photosynthetic rate of PC compared with WT were increased by 13.10%(P<0.05)and 29.29%(P<0.05),respectively in the 14 d and 28 d after flowering under low nitrogen conditions.At the same time,under low concentration of nitrogen,relative to untransformed wild type rice,activities of Rubisco carboxylase of PC were increased by 67.86%and 52.63%(P<0.05)in the 14 d and 28 d after flowering respectively,while activity of nitrate reductase of PC were increased 79.49%(P<0.05)and 17.96%.Meanwhile,activity of glutamine synthetase of PC was increased 28.48%(P<0.05)only in the 28 days after flowering comparing with WT.In order to tolerant to low nitrogen condition,PC can increase different enzymes activity of nitrogen metabolism at different stage.3.In order to further explore the mechanism of PC tolerance to low nitrogen condition,we studied the activities of PEPC,NR,glycolate oxidase and glycerol kinase at low nitrogen in hydroponic for 7 days.The activities of PEPC,NR of PC relative to WT significantly were increased(P<0.05),glycolate oxidase and glycerol kinase activity were significantly increased compared with WT(P<0.05).Relative to WT,the expression of photorespiration related genes OsPGLP,OsGLO5 and OsGLYK of PC were reduced under normal nitrogen condition(P<0.05).However,under low nitrogen condition the OsGLO5 gene of PC was upregulated(P<0.05),but OsPGLP and OsGLYK genes downregulated(P<0.05),but the expression of these three genes were higher than WT(P<0.05).The genes expression related to photorespiration of PC under normal condition were less than WT,indicating that introduced PEPC gene might reduce photorespiration of rice.Meanwhile,under low nitrogen condition,the genes and enzymes related to photorespiration of PC were higher than WT,indicating that improving photorespiration may be one of ways for PC to be tolerant to low nitrogen stress.In a word,transgenic rice with high expression of maize C4-PEPC can increase the activities of some enzymes related to carbon and nitrogen metabolism,and some genes espression and the acti-vities of enzymes related to photorespiration as well,under low nitrogen conditions,which be helpful to resist low nitrogen stress.