Bioinformatic And Functional Analysis Of Mulberry Genes Involved In Lignin Biosynthesis

The content of lignin is next to cellulose, which consists of16%-30%in plants. It plays an important role in mechanical strength, water transportation and microorganism denfense. The presense of lignin is one of the important evolution characteristic adapted to land environment. However, the existence of lignin can increase the cost of paper-making, reduce the digestibility of forage, and constraint of the production of bioethanol, which has serious effect on the utilization of palnt resources.Mulberry is one of the perennial woody plants widely used for sericulture. It produces a lot of branches every year which is the most abundant by-product resources during the sericulture production process. But the utilization of mulberry branches is very low. As a result of reduction of coal oil and other non-renewable resources, bioethanol becomes the best alternative energy. The usage of food crops as raw material has scale limitation and sustainability. If we can use mulberry branches as material to produce bioethanol, will not only improve the utilization of mulberry resources but also alleviate the shortage of energy problems in some extent. Therefore the study of mulberry genes involved in lignin biosynthesis, analysis the mechanism of lignin formation, cloning the key genes in lignin biosynthesis, and changing the lignin content or lignin composition to promote bioethanol production by genetic engineering are very important in the utilization of mulberry resources. In the present research, we obtainced65lignin biosynthesis related genes through informatic analysis. Two genes MnC3H and MnF5H were inverstigated. cloned and transformed to tobacco. The results are as follows:1. Bioinformatic analysis of lignin-related genes in mulberryThrough bioinformatic analysis we obtained65lignin related genes from the mulberry genome database including seven PAL genes, three C4H genes, thirteen4CL/4CL-like genes, six HCT genes, eight CCR/CCR-like genes, three CoAOMT/CoAOMT-like genes, eight CAD genes, two F5H genes, and fourteen COMT/COMT-like genes. It revealed that most of these genes have the domains conserved in each protein family. The majority of these genes were highly expressed in roots, indicating that these genes might be directly involved in the biosynthesis of lignin. We found that the promoter of these genes have conserved AC element which was considered to be responsibe for the specific expression in xylem. Besides AC element, the promoter resgions of these genes have other elements including hormone response and stress induced, suggesting that these genes not only participate in lignin biosynthesis but also play important roles in the biotic and abiotic stess in mulberry.2. Expression analysis of key lignin biosynthesis genes in different mulberry speciesWe investigated the expression of key lignin biosynthesis genes in the stems and leaves of different mulberry species (Morus alba var. multicaulis, Morus alba var. pendula Dippel, Morus.atropurputea Roxb, Morus mongolica C. K. Schneid, Morus alba Linn) by real-time PCR. The results indicated that all of these genes were highly expressed in stems with the highest in M.alba var.pendula Dippel, the second in M.mongolica C. K. Schneid (except C3H、4CL2and F5H). However the expression of these genes in the stems and leaves of M. alba Linn was very low.3. Gene cloning of MnC3H and MnF5H and the genetic transformation of tobaccoIn order to study the function of MnC3H and MnF5H in mulberry, we cloned these two genes and constructed the overexpression vector pLGNL-35S-MnC3H and pLGNL-35S-MMNF5H. The resultant plasmids were transformated into tobacco. Our results showed that overexpression of these two genes have no side effect on the growth of tobacco. And the overexpression of MnC3H led to the incease of lignin content, but had different effection on the biosynthesis of S lignin and G lignin in tobacco stems, indicating that MnC3H can promote the synthesis of G lignin but inhibit the synthesis of S lignin. The overexpression of MnF5H in tobacco led to the increase of S lignin but the decrease of G lignin, but the total content of lignin was basically unchanged. The increasing biosynthesis of S lignin can decrease the cost of production of bioethanol, which plays an important role in improving the utilization of mulberry.