Cloning Of Sbpase Gene In Mulberry And Effect Of Yellow Dwarf Disease On Its Expression

Mulberry yellow dwarf disease caused by phytoplasma is a main disease in mulberry and restricted the development of silkworm. Methods of spreading and rules of happening of phytoplasma have been realized, but the molecular mechanism of disease has not made material evolvement because phytoplasma couldn't bring up by manual work at present. So it has bacome especially important that SBPase resisted the infection of mulberry yellow dwarf disease.The enzyme sedoheptulose-1,7-bisphosphatase(SBPase) which was the key regulator in the Calvin cycle and played the particular roles in regulating carbon partioning to starch and sucrose. The function of SBPase was important to accumulation of alimentation in plant leaves. If the gene sbpase was expressed effectly in mulberry, it would develop the efficiency of photosynthesis and enhance the quality of mulberry leaves. So it would restrain diseases of mulberry in some degrees, and alleviate the damage of the diseases in silkworm.The material of this study were mulberry leaves and cloned the full-length sbpase cDNA of mulberry by Reverse transcription PCR( RT-PCR ) and Rapid amplification of the cDNA ends (RACE) PCR and analyzed this sequence. This paper also studied the changes of expression of sbpase and the photosynthetic characteristics in mulberry leaves infected by yellow dwarf disease. The main results were as follows:1.The material of this study were mulberry leaves. The results showed that total RNA was extracted by means of imprived hexad cetyltrimethylammonium bromide (CTAB) method was good and used in the following experiments such as molecule cloning.2. Degenerate PCR primers were designed according to the most strongly conserved domain according to SBPase polypeptides of published. The fragment was cloned by RT-PCR and the bands of the expected size was 530 bp. Sequence comparisons by performing Blast Search in GenBank database ( http://www.ncbi.nlm.nih.gov/BLAST/ ) analysis revealed that the fragment had high homology with many other plant SBPases. RACE was used to amplify the 3'-and 5'-end of the SBPase cDNA and obtained two fragments which size were 540 bp and 600 bp. Sequence comparisons by performing Blast Search in GenBank database analysis revealed that Both fragments were the sequences of 3'-and 5'-end of sbpase.3. By comparing the overlapping sequences of partial cDNA fragments, 3' and 5'-PCR products, the full-length cDNA sequence of sbpase was deduced. The 1527 bp cDNA contains a 1179 bp open reading frame which begins at the translation inition ATG codon and ends at the TAA termination codon. This cDNA was deduced to encode a peptide of 393 amino acids and it was named Msbpase. The nucleotide sequence data reported in this paper will appear in the GeneBank databases under accession number DQ995346. Sequence comparisons by performing Blast Search in GenBank database and multiple alignment analysis revealed that the sbpase of mulberry had high homology with many other plant sbpases such as Arabidopsis thaliana, Spinacia oleracea and Oryza sativa.4. The 3-D structure of SBPase was determined by predicting of SwissProt. Molecular modeling showed that SBPase obtained two identical subunits and each subunit is composed of a series of alpha helices and beta sheets joined by turns and loops. Each subunit is paired forming a functional homodimeric with a disulphide bond on the interface between the subunits.5. The total RNA were extracted from the infected leaves of infected trees, the healthy leaves of infected trees and the healthy leaves of healthy trees, then the RNA was transferred to a Hybond nylon membrane filter. Using sbpase cDNA as probe, Northern blot analysis proved that the expression of sbpase was down-regulated in the leaves of infected mulberry.6. When the mulberry was infected by phytoplasma, its photosynthesis was restrained significantly and the chlorophyll content, net photosynthetic rate(Pn), stomatal conductance(Gs), transpiration rate(Tr), carboxylation efficiency(CE) and intercellular CO₂ concentration(c) of the leaves of infected trees were declined in comparison with that of the healthy trees, however, the starch content of the leaves of infected trees was increased.