Molecular Characteristics Of Tomato Yellow Leaf Curl Virus In Hebei Province And Its Infectious Clone Constructing

Tomato yellow leaf curl disease is one of the main diseases of tomato in the world. The typical symptoms of the diseased plant were leaf curl, crimple, yellow, plant dwarf and so on. Recent years, the disease broke out in Guangdong, Guangxi, Shanghai, Jiangsu, Zhejiang, Shandong, Henan and Anhui. In the autumn of 2009, it widely occurred in tomato-produced area of Hebei province and made significant loss on the tomato production.The isolates of tomato yellow leaf curl virus isolated from Langfang,Gaocheng,Weixian, Hebei province has been cloned and sequenced, and the infectious clones of the isolate L2 were constructed in this study. The results were as follows:The diseased tomato samples exhibiting yellow and leaf curl were detected by PCR with the degenerate primer AV494 and CoPR of Begomovirus in Geminiviridae. The result showed that the specific 570bp-fragment was amplified from all the samples. These indicated that the diseased tomato samples from Langfang city of Hebei were infected by begomoviruses. According to the sequence of 570bp fragment, a pair of adjacent primer was designed to amplify full-length DN A-A sequence of the isolate L2, the cloning and sequencing results showed that L2 DNA-A contains 2781 nucleotides (nt) and encodes six potential open reading frame (ORF), with two (AV1 and AV2) in virus-sense and four (AC1, AC2, AC3 and AC4) in complementary sense. The intergenic region (IR) has a stem-loop structure with 11nt stem and 11nt loop, and conserved sequence TAATATTAC between the AV2 and AC1. Sequence comparison results showed that the sequence identities of the isolate L2 with isolates of Tomato yellow leaf curl virus (TYLCV) were more than 94%, and L2 shared the highest identity with TYLCV-IL DNA-A at 99.68%. No component B (DNA-B) and satellite beta molecular (DNAβ) were found.Using the degenerate primer SSPF1 and SSPR1 of begomoviruses, the diseased tomato samples exhibiting yellow and leaf curl symptoms were detected. The PCR result showed that the expecting 660bp-fragment was amplified from all the samples. These indicated that the diseased tomato samples from Gaocheng city of Hebei were infected by begomoviruses. According to the sequence of 660bp fragment, a pair of adjacent primer was designed to amplify full-length DNA-A sequence of the isolate GT. The cloning and sequencing results showed that GT DNA-A contains 2781 nt and encodes six potential ORFs, with two (AV1 and AV2) in virus-sense and four (AC1, AC2, AC3 and AC4) in complementary sense. The IR has a stem-loop structure with 11nt stem and 11nt loop, and conserved sequence TAATATTAC between the AV2 and AC1. Sequence comparison results showed that the sequence identities of the isolate GT with isolates of Tomato yellow leaf curl virus (TYLCV) were more than 94%, and GT shared the highest identity with TYLCV-IL DNA-A at 99.53%. No DNA-B and DNA p were found.The four isolates of tomato yellow leaf curl virus from Weixian of Handan city, Hebei province were cloned using rolling circle amplication (RCA). The total DNA was extracted from the diseased tomato tissues infected by isolates HI, H2, H3 and H4. The begomoviruses genomes were amplified by RCA. The RCA products were digested with restriction enzymes BamH I, EcoRⅠ, SacⅠ, XbaⅠand EcoRⅣ. A single fragment with 2.7kb size was got using EcoR I digest, and the fragments were cloned. The sequencing result showed that all DNA-A of isolates H1, H2, H3 and H4 were 2781 nt. The four isolates DNA-A shared 99.6%-99.9% sequence identity. They have same genome structure, and all encodes six ORFs, with AV1 and AV2 in virus sense, AC1, AC2, AC3 and AC4 in the complementary sense. The IR has a stem-loop structure with 11 nt stem and 11 nt loop, and conserved sequence TAATATTAC between AV2 and AC1 Sequence comparison results showed that all the sequence identities of the four isolate with isolates of Tomato yellow leaf curl virus (TYLCV) were more than 94%, and they shared the highest identity with TYLCV-IL DNA-A at 99.84%. No DNA-B and DNA (3 were found too.Phylogenetic analysis based on the DNA-A sequences indicated that the virus isolates in Hebei province were most closely related to the isolates of TYLCV-IL, and clustered together to form a branch. Therefore, the begomoviruses infected tomato in Hebei should belong to mono-component TYLCV strain Israel (TYLCV-IL), and the genetic variation among DNA-A of the isolates were very small.In order to further study the pathogenicity of TYLCV, the infectious clone of TYLCV-[L2] pGreenⅡ0000-2.OA was constructed by gene cloning technique. The results of Agrobacterium infiltrating Nicotiana benthamiana indicated that the inoculated plants showed yellow, leaf crimple and interior leaves curl 12 dpi; the virus isolate L2 were detected from the DNA of the leaf tissues exhibiting symptoms by PCR. The above results suggested that the pGreen 110000 -2.0A had infectivity. The study laid the foundation for the virus pathogenicity, gene function, infection circle and control technology.