Cloning And Expression Analysis Of Phosphoenolpyruvate Carboxylase Gene Family From Cassava

Phosphoenolpyruvate carboxylase（PEPC）is a key enzyme in C₄ plant photosynthesis.In C₄crops,it is mainly responsible for the initial fixation of CO₂ and catalytic carbon assimilation.In addition,it is also involved in regulating guard cells,promoting Rhizobium nitrogen fixation,seed germination,and plays an important role in plant stress resistance.Cassava is a C₃-C₄ plant and an important food crop in the tropics.It has the characteristics of high light efficiency,high starch accumulation and high yield.In this paper,the MePEPC family of cassava was studied by bioinformatics,q RT-PCR,homologous cloning and cassava genetic transformation.The specific results are as follows.1.The results of bioinformatics analysis showed that there were five members in the cassava PEPC gene family:MePEPC1,MePEPC2,MePEPC3,MePEPC4 and MePEPC5,which were distributed on chromosomes 2,3,6,14 and 15 respectively;Subcellular localization predicted that all five members were located in the cytoplasm.The results of phylogenetic tree showed that MePEPC1,MePEPC2 and MePEPC5 were plant proteins,MePEPC3 and MePEPC4 were bacterial proteins.The prediction of secondary structure and tertiary structure showed that in MePEPCs family,the proportion of alpha helix is 55.88%～62.71%,and the proportion of irregular curl is 27.56%～33.43%.Domain analysis showed that the distribution of conserved domains of all MePEPCs members was similar.The promoter region of MePEPCs contains different numbers of light response elements,hormone response elements and stress response elements.In particular,the ABA response element ABRE exists in the promoters of all genes.2.The full-length sequences of five members of MePEPCs were obtained by PCR using the c DNA of cassava cv.SC8 as the template.Among them,MePEPC2 has alternative splicing at the fourth intron,resulted in premature termination of translation and loss of function.3.q RT-PCR was used to analyze the expression of MePEPCs members in different tissue parts.The results showed that MePEPC1 was mainly expressed in leaves,MePEPC4 was expressed in all tissue parts,MePEPC5 was mainly expressed in flowers,and MePEPC2 and MePEPC3 were was almost invisible in all tissue parts.The results of different time point analysis showed that MePEPC1,MePEPC4 and MePEPC5 were regulated by light.The expression level of MePEPC1was increased in leaves and roots under drought treatment.Under ABA stress,the expression of MePEPC1,MePEPC4,MePEPC5 in leaves were decreased,while the expression of MePEPC1was increased.That is,the expression of MePEPC1,MePEPC4 and MePEPC5 are regulated by ABA.4.The highly expressed members MePEPC1,MePEPC4,MePEPC5 were inserted into p CAMBIA1300::GFP vector and injected into tobacco for subcellular localization.The subcellular localization results showed that MePEPC1 and MePEPC5 were located in the cytoplasm,and MePEPC4 was located in the cytoplasm and chloroplast.5.According to the above information,MePEPC4 with high expression in chloroplast was screened to be used to construct RNA interference vector and transformed into friable callus of cassava cv.SC8.20 positive transgenic lines were obtained after PCR identification.The expression analysis showed that the interference effect of MePEPC4 was significant,and the relative expression level of MePEPC4 in MePEPC4i-15 was only 1/18 of that in the control.At the same time,the relative expression levels of Me PPDK and Me SUT1 in the upstream and downstream genes of PEPC metabolic pathway were investigated.It was found that the expression levels of Me PPDK and Me SUT1 were significantly lower than those in the wild type in several lines with significantly decreased expression levels of MePEPC4.The above research provides basic data for in-depth study of the function of MePEPCs in cassava,and provides candidate genes for cassava high light efficiency breeding.