Cloning, Expression And Functional Of HCP1Gene In The Process Of Wheat Infected By Leaf Rust

In the process of wheat leaf rust interaction, incompatible combinations of wheatusually resist leaf rust infection by hypersensitive reaction (HR). In the early stage of HRformed, one of the important physiological and biochemical changes that occur oxidativeburst to produce reactive oxygen species. Our lab has long been engaged in the interactionsmechanism between wheat and leaf rust. We had established a SSH library of the wheatinfected by leaf rust, from which the HCP1(an iron/ascorbate-dependent oxidoreductase)EST was selected. And this EST had a high level of expression in the wheat leaves after theinoculation of incompatibility leaf rust. This result implied that the high expression level ofHCP1might play an important role in the scavenging of H2O2in incompatiblecombination. This study use wheat (Triticum aestivum L.) variety Lovrin10(L10) withleaf rust (Puccinia recondita) physiological races165and260to compose compatible andincompatible combinations respectively. The aim is investigate HCP1, H2O2in the role ofwheat resistance to leaf rust induced HR. In this study, we constructed HCP1geneover-expression and RNAi binary expression vector, then used Agrobacterium mediatedwheat mature embryos to genetic transformation in order to study the function of HCP1and the relationship between HCP1, H2O2and HR. We obtained the following results:1. The HCP1gene was cloned through the method of RT-PCR from the wheatseedlings’ leaves.2. We used the fragment of HCP1which removed the terminator combining with GFPto construct the plant express vector and inject the tobacoo leafs. Through the observationof Confocal, we discovered the expression of HCP1in nucleus and cytoplasm.3. The expression of HCP1in mRNA level at different hour’s post the inoculation ofleaf rust was verified by qRT-PCR. The results showed that: this gene was involved in thedefense response of wheat resistantance to leaf rust infection.4. Using virus induced gene silencing (VIGS) to silent L10HCP1gene, we foundthe wheat increased the accumulation of H2O2and HR extension infected by leaf rustrace260post48h. It indicated HCP1gene was important to H2O2scavenging in plant.The result showed that H2O2might have positive regulation of HR. 5. We constructed the HCP1over-expression and RNAi vector and usedtransformation system of wheat mature embryos to transform wheat varieties5389and L10.We acquired the PCR positive plants.In summary, HCP1as an iron/ascorbate-dependent oxidoreductase, In the interaction,HCP1played an important role in the scavenge of H2O2and produce a series of defensiveresponses.This research helps us to better understand the role of H2O2in the plant immuneresponse.