Cloning Of CDNAs Induced By Soybean Cyst Nematode Race 4 From Infected Soybean Roots

The soybean cyst nematode (SCN), Heterodera glycines Iehinohe, is one kind of soil sedentary endoparasitic nematodes and one of the most important parasites of soybean. SCN is a very harmful soybean pathogen, which is very difficult to control, because of extensive distribution, severe harm, broad host and long period surviral and so on. Although the application of resistant varieties can decrease the damage from SCN, the yield of soybean could be still decrease. It is best solution to use resistant varieties to decrease the damage from SCN. But the resistant varieties will often lose the resistant to SCN with the alternation of SCN race. Therefore, it is necessary to understand the interaction between SCN and soybean. The second-stage juvenile of SCN penetrates soybean roots and causes the formation of specialized feeding cells which are usually called syncytium in the roots?vascular system. The morphological and physiological change of syncytium is considered to be the result of soybean gene expression induced by SCN directly. However, the mechanism of this change is not very clear. Identifying and characterizing resistant gene is very important to study the interaction between SCN and soybean. Huibuzhiheidou, a local germplasm of Shanxi province, is a strong resistance source to SCN race 4 and was explored in this work. 2-day-old Soybean seedlings were inoculated by J2 of SCN race 4. Differential display of mRNA was used to detect host gene expression changes during the compatible interaction between soybean and SCN. Thirty-six cDNA clones corresponding to mRNAs with different abundances in SCN-infected (19 cDNAs) and uninfected (17 cDNAs) roots were identified. Six of them were recovered as plasmid clones that showed hybridization intensity differences in reverse dot-blotting assay. They are A32 clone(BI173978) from soybean roots five days after inoculation, B12 clone(B1173979) and B71 clone (B1173980) from soybean roots ten days after inoculation, Cli clone(B1173981), CPI2 clone(B1173982) and CP32 clone (B1173983)from soybean roots fifteen days after inoculation. The sequence of above six clones have been registered in GenBank. Accession numbers are showed in the brackets. The sequences of six clones were subjected to data base comparisons with the aid of BLAST algorithm. All of them showed strong similarity to cDNAs which were in soybean EST library constructed by Shoemaker. Especially, A32 clone showed strong similarity to EST eDNA for coding late elongated bypocityl protein, tomato root nutrition deficiency cDNA and tomato cDNAof pseudomonas resistance. We conclude initially that A32 is a differential display cDNA induced by SCN race 4 from infected soybean roots and related to resistance in soybean. Based on the sequence of A32 clone, two Gene Special Primers (GSP1 and GSP2) were designed for RACE method to amplify the nucleic acid sequence between A32 and unknown sequence at the 5?end of the mRNA. Three unknown fragments were amplified from RT-PCR reaction and two of them were recovered successfully. The result of sequencing showed that both of them were unknown sequences between A32 and the 5?end of mRNA. A new cDNA sequence (523bp), named A32-523, was generated by combining 5扲ACE product and the sequence of A32 clone. With the aid of BLAST algorithm, the nucleotide sequence of A32-523 showed 87.3% similarity to an unknown EST cDNA (GenBank accession no.: BF066505) in soybean EST library constructed by Shoemaker. The nucleotide sequence of A32...