Cloning And Sequence Analyzing The Gene Of Buckwheat Proteinase Inhibitor

Insect pest is one of the major causes for the crop reduction worldwide. Every year, the total economic loss resulted from crop pest amounts to nearly thousands of billion dollars. Many adverse effects such as ecological damage, environmental pollution and insecticide resistance are all the consequences of constant using of chemical insecticides. On the other hand, natural bio-insecticides haven't got general application due to its high cost, field instability and narrow anti-insect spectrum. To reduce insect damages as much as possible, traditional genetic crossbreed is a possible way to cultivate anti-insect crops, but the time-consuming selective process and lack of perfect parent plants are unavoidable problems. A newly effective way has been produced through gene engineering technology and till now, above ten kinds of relative anti-insect genes respectively from animals, plants and microbes are available for further gene transplantation. Among them, proteinase inhibitor (PI) is an important one with particular qualities and effects in anti-insect gene engineering.Buckwheat belongs to Polygonaceae family. We extracted RNA respectively from buckwheat leaves and cotyledons and got the cDNA by reverse transcription. The following accomplishments were achieved using gene cloning and screening cDNA library methods.1. According to the reported amino acid sequence of PI from common buckwheat and other kinds of plants, special primers were designed. Using the cDNA fragments as the templates, part of the structure gene sequence encoding BPI was amplified through RT-PCR. It is an ORF including two gene fragments of 158bp and 84bp which respectively encode 52 and 27 amino acids. Comparing with the reported amino acid sequence in BLAST database, they have 51% and 100% homology with buckwheat proteinase inhibitors, and comparing with proteinase inhibitors from other plants, the homology arrives at 57% and 74%.2. To get the full gene sequence of BPI, part of 3'-end gene sequence was also cloned with particularly designed primers by 3'-RACE PCR combined with nested PCR. Now the total amplified DNA fragment contains altogether 385bp including a translation region of 183bp encoding 61 amino acids and a untranslated region of 202bp rich in 70% AT. This is the first report worldwide about the cloned gene sequence of BPI.3. In addition, a cDNA library with a liter of 4.8 X 109pfu/ml from buckwheat seeds was also constructed so as to get the complete BPI gene sequence. Screening the cDNA library using dig-dNTP-marked aim DNA sequence as the random primers may bring out ideal gene sequence, which is helpful for the following gene transplantation.