The Expression Analysis Of Important Genes In Response To P Deficiency And Sequence Variation Os PAPP Gene Based On The SSH Library In Maize

Phosphorus is the important component of nucleic acids, the nuclear protein, the phosphatide, ATP and so on. It is one of the most macronutrients in plant growth process and plays an important role in plant metabolism. But plants can only absorb inorganic phosphorus, and organic phosphorus must be converted into inorganic phosphorus to be absorbed by plants. Eighty percent of phosphorus in soils is immovable and can not be absorbed by plants. In order to meet the normal growth of crops, addition of phosphorus fertilizer is a usual method to ensure normal growth and development of crops for a long time, but global resources of phosphate rock will disappear in the end of this centuryCrop breeding for tolerance to low phosphorus is very urgent. In China, maize is the largest in planting area and the second in total production. It is important for cultivating maize Pi-tolerant variety by researching of molecular mechanism of maize in phosphorus stress and identifying the fuction of related genes response to low phosphorus.This research was based on SSH library of maize root phosphorus stress, We select two kinds of genes which belong to the phosphorus cycle and signal transduction through EST sequencing and homology comparison analysis. There are PAPP(Purple acid phosphatase precursor), PAP(Purple acid phosphatase), AP(Auxin-inducible protein), A PY(Apyrase), CIPK(Calcineurin B-like protein interacting protein kinase), AAPT(Aminoalcohol-Phosphotransferase), STPK(Serine/threonine kinase protein),7 genes totally. Gene expression of these 7 genes were are performed using real time quantitative PCR in roots and leaves of inbred lines 178 and 9782 dealing with low P and normal P to verify the transcription level, and the part of PAPP sequence in 14 maize inbred lineswere analyzed by fragment length polymorphism and SNP analysis. The main results are as follows:1:Under low phosphorus stress, PAPP encodes 457 amino acids with a trans-membrane structure. The PAPP in maize and in Ricinus communis are in the same evolutionary branch by analysis of MGEA. The change of expression level of PAPP in roots is more than in leaves. It is down-regulated expression in the roots and up-regulated expression in leaves of 178 and 9782. The gene is induced up-regulated expression originally, especially in the roots of 178. The expression quantity is highest in 24h. The gene is induced down-regulated expression originally in 9782.PAP encodes 349 amino acids with one trans-membrane structure. The PAP in maize is in the same evolutionary branch with PAP 4 and PAP 17 in Arabidopsis thaliana by analysis of MGEA. The characterstics of gene expression for PAP is that it is distinctly up-regulated expression in leaves and down-regulated expression in roots in 72h. Moreover, the expression pattern is the same between 178 and 9782 in 72h. The expression quantity is highest in 12h in roots and in 72h in leaves.AP encodes 105 amino acids with two predicted trans-membrane structures. The expression analysis is that expression quantity in roots is higher than in leaves. It is indicated that the gene has organization differential expression and expression in roots concentrately.APY encodes 176 amino acids without trans-membrane structure. The APY in maize is in the same evolutionary branch with Phaseolus vulgaris, Ixeris repens, Medicago truncatula by analysis of MGEA. The expression quantity of APY in roots is higher than in leaves. The expression quantity of APY is very low in leaves of 9782 and almost can not detect. It is worth to study further. There is no difference of expression quantity between roots and leaves at 6h and 72h, respectively, in.CIPK encodes 449 amino acids without trans-membrane structure. The CIPK in maize shares the same evolutionary branch with Ricinus communis by analysis of MGEA. The expression analysis of CIPK shows that there is apparent down-regulated dot in 24h, the molecular characteristic of this gene needs further research under low phosphorus stress.AAPT encodes 757 amino acids with 8 trans-membrane structures. The AAPT in maize shares the same evolutionary branch with Sorghum bicolor by analysis of MGEA. In the treat of 12h, the expression quantity of AAPT is the highest and it is up-regulated in roots. It is not obvious in latest induced expression. The change of expression of AAPT gene isn't obivious in leaves.STPK encodes 341 amino acids without trans-membrane structure. In the treat of 12h, the expression quantity of STPK is the highest. It is up-regulated in roots and down-regulated in leaves. At 72h, expression quantity of STPK is the highest in leaves.2:PAPP is a single copy in maize with 5 exons and 4 introns by analysis in Maizegdb web. Primers of the exon region of PAPP were designed to select primer of ppl which is in the second region of exon. The primer ran out of length polymorphism differences in 14 inbred lines [(CML-473)-B, Cheng698, B73, Dan599, Chang7-2, S37, Dan598,975-12, Huo Tang Huang, RP125,18-599, R08,178,9782]. Fourteen inbred lines of PAPP gene fragment were multiple sequence alignment analysised in the CLUSTALW. We come into conclution that there are the same sequence band in eight inbred lines (Cheng698, Dan599, Chang7-2, S37,975-12, Huo Tang Huang, R08,178) and in six inbred lines [(CML-473)-B, B73, Dan598, RP125,18-599,9782], respectively. Amplified fragment of PAPP is relatively conservative, and two indels and five SNP locis were found. The two indels were constructed by 5nt and 7nt, respectively. The polymerism locis account 7.76% of cDNA sequence. In the 1394nt of Huo Tang Huang, C mutations into the T with single base and it's a non-synonymous mutation loci only, so that amino acid of alanine(Ala) mutations into valine(Val). In the 147lnt of Huo Tang Huang, T mutations into C with single base. In the 1395nt of S37 and 178, A mutations into G with single base. In the 1458nt of Chang7-2. A mutations into T with single base, respectively. More importantly, compared to those 8 inbred lines sharing similar sequencewith 178,6 inbred lines sharing similar sequence with 9782 have more inserted 5nt(TGTTG) between 1541nt and 1545nt.