Cloning And Dynamic Expression Analysis Of Differential Expression Genes In Pyrus Pyrifolia Induced By Thermotherapy

Thermotherapy is used widely to eliminate viruses from fruit trees. Previous studies mainly focused on elimination of viruses from hosts. However it is rarely understood on the interactions between hosts and virus during thermotherapy. Previously, two suppression subtractive libraries were constructed from in vitro pear(pyrus pyrifolia) under heat treated at 37℃and 149 ESTs were screened. In this study, the relationship of ASGV titers and the dynamic expression of high molecular weight heat shock protein gene (HSP70), small molecular weight heat shock protein gene (smHSP), photosystem II light-harvesting protein gene(LHCII) in the forwand library and chitinase (EC), metallothionein (MT) genes in the reverse library in in vitro pear plants during thermotherapy were further analyzed,which will be helpful to know the relevance between differential expression and viruses titers changes,and provide the reference gene for study the mechanism of viruses elimination from hosts. Full-length DNA and cDNA sequences of HSP and EC genes were cloned and analyzed to clarify their molecular sequence characteristics,which could provide meterials for further function study of HSP and EC funtion. Seeking results are listed as followings:1. The relationship of the dynamic expression quantity of HSP70, LHCII, smHSP, EC, MT and ASGV titers in Pyrus pyrifolia "huanghua" plant shoots during with the periods of heat treatment were analyzed by Real-time RT-PCR and RT-PCR. The results showed that thermotherapy induced the expression quantity of HSP70, LHCII and smHSP genes, in which, smHSP gene was non-constitutive expression. The expression quantity of genes increased from 1d to 10d, and reached at the highest point at 10d,while decreased evidently after 15d. No signal from these genes was detected at 35d. The expression quantity of EC and MT genes were obviously decreased from Id to 35d during thermotherapy and reached the lowest point at 35d. The titers of ASGV in Pyrus pyrifolia decreased significantly from 1d to 10d, and no ASGV signal was detected from 10d to 35d. Based on the analysis of the relationship of the dynamic expression quantity of genes and ASGV titers, it is deduced that high temperature may induce or activate the over-expression of HSP70, LHCII and smHSP genes which were related to viral resistance in the hosts, which inhibited viral replication and movement in host plant.2. The sequences from HSP70 and EC genes in Pyrus pyrifolia "huanghua" plant shoots were cloned and analyzed. The results showed that the full-length sequence of 1950 nucleotides(nt) from HSP and 1053 nt from EC were obtained.encoded 650 amino acids(aa) and 334 aa respectively. The Blast search results showed the highest sequence identity(98.8%) at aa level of full-length sequence from HSP70 with that of the apple(Genbank accession No. AF161180.1), and 86.9% at aa level of full-length sequence from EC with that of the pear host(Genbank accession No. FJ589784). The results will provide materials for further study of HSP and EC protein. It is deduced from sequence analysis that HSP gene belonged the family "HSC70"...