Expression Analysis Of Polygalacturonases Genes In Whitepearmain Apple Fruit And Construction Of Antisense Eukaryotic Expression Vector

Fruit firmness is an important index of fruit taste and preservation time, it is closely related to the resistant ability of mechanical damage,fruit cracking and fungal infection. The degradation of pectic substance and cell separation result in fruit softening. In this process,Polygalacturonases (PGs) play an important role. This enzyme,which acts as a key enzyme to change the structure of cell wall , can catalytic the decomposition ofα- (1.4) polygalacturonic acid and then lead to fruit softening in the end. 1-MCP was found as a new inhibitor of ethylene recently,it can competitively bind to ethylene receptor to inhibit the combination of receptor and ethylene. The inhibition delays or prevents plant physiological aging,and was widely used in the fields of cut-flower preservation and fruit preservation recently.For study the relationship of fruit firmness,ethylene production,respiration rate and expression of PG gene in cold-storage apple fruit,the improved CTAB method was used to isolate the total RNA and the method of reverse transformation polymerase chain reaction (RT-PCR) to clone the PG gene. And also,1-MCP was chose as the inhibitor of ethylene, and the expression of PG gene was studied by Northern blot analysis. Furthermore, the antisense expression vector have been constructed and transformed it into tobacco. The main results are as follows:1.High-quality total RNA sample was obtained from apple fruit by the improved CTAB Method.2.The PG gene was cloned by RT-PCR from the apple peel.3.The relationship among the expression of PG gene,ethylene and fruit firmness was studied in this research. It showed that 1-MCP inhibited expression of PG gene, respiration rate, and ethylene production which increased gradually in the control fruit with the delayed storage time, and significantly decreased the softening rate with the percentage of 8.7% while 51% in the control fruit after 13 days at ambient room temperature. It was proved that PG was the key enzyme controlling the fruit softness, and induced by ethylene.4.The antisense expression vector was conducted and transformed into tobacco and the transgenic plant was obtained.