Isolation And Function Analysis Of Pepper Root-Knot Nematode Resistant Gene CaMe And Defense Gene CaSn

Root-knot nematodes (Meloidogyne spp.) are rootendoparasites of numerous crops worldwide and are the most damaging nematode pest in agriculture. Solanaceae vegetable, especially pepper and tomatoe are the most-widely cultivated in the world (FAO1996), and they are susceptible to root-knot nematodes (RKN).Several root-knot nematode resistance genes have been discovered in different pepper (Capsium annuum L.) lines; however, little of them have yet been cloned. The pepper HDA149is a DH (doubled-haploid) line, which has a single dominant gene Me3, confers heat-stable resistance to root-knot nematodes (RKN). Using SSH, we got an EST fragment of nematode resistant gene from the pepper HDA149line induced by RKN. In addition, base on the nucleotide sequence, gene CaMe was obtained through RACE amplification. The full-length cDNA of CaMe was4,697bp, which included a putative transcription start site, a potential open reading frame of3,600bp and a poly (A) tail.The potential open reading frame encoded for1,199amino acid residues and contained a potential nucleotide binding site (NBS) domain and a leucine-rich repeat (LRR) domain. RT-PCR show that CaMe gene was highly expressed in root, young leave, and expressed at a lower level in stem, flower and young fruit. Expression profiling analysis revealed that CaMe gene was begin to expressed in root after RKN inoculation6h, the highly expressed at12, and lowly expressed after72h. In the HDA149root tip, it can be observed that the tissue necrosis or hypersensitive response was triggered at the site of root-knot nematode invasion. We targeted CaMe for degradation with tobacco (Nicotiana tabacum) rattle virus (TRV)-based virus-induced gene silencing using Agrobacterium infiltration with a TRV-CaMe-1/2construct. CaMe-1and CaMe-2were the motif located in conserve and special domain of CaMe cDNA respectively. In most HDA149plants infiltrated with the TRV-CaMe construct, the root-knot and mass were increased in the most conspicuously level than control, the pepper plant transform its resistance to susceptibility. These indicating that the RKN resistance is mediated by the gene CaMe.The pepper Carolina Wonder was a nematode-resistance line. Through SSH, we got an EST fragment of Snakin gene from the pepper Carolina Wonder line induced by RKN. In addition, based on the nucleotide sequence, gene CaSn was obtained through RACE amplification. The full-length cDNA of CaSn was655bp long, which included a putative transcription start site, a potential open reading frame of315bp and a poly (A) tail. The potential ORF encoded for104amino acid residues, which contained a potential signal peptide (SP), acidic sequence and mature protein, in addition, the12Cys were in disulphide form. It was predicted that the mature protein was secreted and belong to the Snakin/GASA subfamily Ⅱ. Through the prokaryotic expression system, the protein CaSnakin encoded by CaSn was be expressed in E.coli. The RT-PCR show that CaSn gene was the highest expressed in shoot apex, then the root tip and young leaf, bur expressed at a lower level in stem, flower and fruit. Expression profiling analysis revealed that Casn gene was begin to expressed in root after RKN inoculation6h, the highly expressed at12, and keep expressed still after72h. With tobacco rattle virus (TRV) based virus-induced gene silencing using Agrobacterium infiltration with TRV-CaSn construct, it was show that the root-knot and mass were increased in the most conspicuously level than control in most HDA149plants infiltrated with the TRV-CaSn construct. These indicating that CaSn gene was take part in the defence to nematode invasion.