Construction Of SSH CDNA Library From Ning RS-1 And Cloning The PGIP Gene And Its Expression Vector Constructing

Oilseed rape is one of the four major oil crops in the world and oilseed rape oil is of the major edible oil in China. Stem rot disease(Sclerotinia sclerotiorum) is a major disease of oilseed rape, which caused by Sclerotinia sclerotiorum usually resulting in 5% of seed yield loss in general year and 30% or even more loss in the year with crop being seriously infected. So far the gene resistance to Sclerotinia sclerotiorum has not been found in Cruciferae as well as its related wild allies that is why there aren't any oilseed rape cultivars with resistance to Sclerotinia sclerotiorum being bred. Obviously to develop a Sclerotinia sclerotiorum-free cultivar is not easy because of the lack of resistant gene. Undoubtely it's very meaningful to search a resistant gene for the prevention of Sclerotinia sclerotiorum disease in oilseed rape.Sclerotinia is a fungal disease. First step of fungal infecting a plant is to secrete a poly-galacturonic acid enzyme (PG) which can degrade plant cell wall and make other plant cell wall degrading enzymes to attack its substrate more easily and provide sugar source for fungal growth and developing. Poly-galacturonic acid-inhibiting protein (PGIP) is able to specifically recognize and inhibit the activity of PG secreted from fungal and to stop the infection of fungal.Ning RS-1, a Brassica napus line with tolerance to Sclerotinia sclerotiorum was used in this study as plant material. The fungal of Sclerotinia used in this research was collected from oilseed rape production fields in Nanjing. A suppression subtractive cDNA library was constructed after Ning RS-1 induced by Sclerotinia sclerotiorum. And the BnPGIP gene cloned from Ning RS-1 was used to construct the BnPGIP-pCABIMA constitutive expression vector. Preliminary analysis was conducted by the method of semi-quantitative PCR. Results are as follows:1. RN A was extracted respectively from the leaves of the normal Ning RS-1 and Ning RS-1 induced by Sclerotinia sclerotiorum and their mRNA was harvested by means of purification. Then the mRNA was transcripted into cDNA by RT. A connector was added on to the cDNA by enzymatic hydrolysis and then two time hybrid was conducted. Finally differential expression of suppression subtracted cDNA library of the induced Ning RS-1 was built.In which the subtractive was used as a tester from the induced Ning RS-1cDNA and the normal Ning RS-1 cDN A was used as a driver. Reverse hybrid library did the opposite. The library built was characterized with a recombination rate of 95% and a base capacity of 2900 indicating the quality of the library was good enough to meet the requirments of screening resistance genes to Sclerotiorum. Several genes related to metabolism of resistance to the disease was identified by sequencing of the clones picked randomly from the cDNA library, such as r-glutamyl cysteine enzyme gene, anti-fungal protein, iron protein, ACC oxidase, and so on.2. A BnPGIP gene from the Ning RS-1 genome was cloned into a DNA sequence by PCR using the degenerate primers designed based on a known PGIP1 sequence in oilseed rape. Amino acid sequence of the BnPGIP gene cloned shared 98% identity with PGIP1. The DNA sequence of PGIP contains an intron and two exons with a coding sequence length of 984bp and encoding 332 amino acids. Finally a constitutive expression vector of Ning RS-1 PGIP-pCABIMA was constructed3. The difference of expression of the BnPGIP gene expressed in normal Ning RS-1 and the induced Ning RS-1 was analyzed by semi-quantitative RT-PCR using translation elongation factor EF-la as an internal control. It was found that the quantity of expression of the cloned gene in the induced Ning RS-1 was significantly higher than in normal Ning RS-1 indicating that expression of the BnPGIP gene inside Ning RS-1 was induced after infection of Sclerotinia sclerotiorum. It could be evidence of improving resistance of Sclerotinia sclerotiorum for oilseed rape by transferring PGIP gene in to it.