Molecular Mechanism Of Different Rooting Capacity Between Two Clones Of Larix Leptolepis×Larix Olgensis

Larix is one of the important tree species in North China and it has highly economic values. The main methods for Larix breeding is still cutting which can get large amount offspring quickly and the offspring has steady genetic background. But Larix is not easy to rooting by cutting method and has significant different among clones. In molecular level, do the research about the genes which related to rooting has big significance. It can explore the rooting mechanism about Larix cutting and it may also improve the rooting efficiency. In our research, by the SSH method, we successfully constructed the cDNA library between two Larix clones which have difference rooting ability.(1)The results of cutting propagation in Larix indicates callus was found afer 10 days, adventitious root primordium was found after 25 days and root was formated since cutting propagation was done. The rooting rate was from 17.18% to 90.63%. Then, sampling time was maded from 6 hours later to 50 days since the cuttings was treated with IBA.(2)Choose two Larix clones which have significant difference in rooting ability as materials. We select different stages during the rooting process to construct the cDNA library by SSH method. After sequencing and assembling, we get 521 UniEST(272 from positive library and 249 from negative library). Blast with the nr Database, we found 163 UniEST in positive library and 179 UniEST in negative library have homologs in Database. The sequence include the genes related to metabolism, signal pathway, transport, resistance, development and cell structure. Which take about 65.8% space in all the sequence. The result suggests that these genes have the function in rooting process.(3)In all the 521UniESTs, 10 of them are related to the hormones. And 6 of these 10 UniESTs have relationship with auxin, 2 with gibberellin, 1 with ABA and 1 with jasmonic acid. In the 6 UniESTs that related to auxin, 2 of them belong to AUX/IAA Family, 1 belongs to NAC gene family and another 3 are the auxin response genes. The result indicates hormones have important functions on growth of adventitious root and lateral root.(4)As it is a very complex, the formation of adventitious root of Larix cutting include the genes related to signal pathway, Transport, metabolism and cell structure. Beside the genes related to the hormones. The result indicates small GTPases was very important in Signal pathway during formatting of adventitious root of Larix cutting. The small GTPases and ABC were very important in transport during formatting of adventitious root of Larix cutting. The wounding-related compounds may improve the growth of adventitious root in Larix cutting.(5)The 2 IAA genes----IAAOL1 and IAAOL2 expressed in root, stem and leaf of Larix. The 2 genes expressed positively with auxin treat, which means they are auxin response genes. During the different stages of the rooting process, IAAOL1 expressed higher in the clone which have high rooting ability. The result indicates IAAOL1 has positive relationship with the rooting ability and it may improve the growth of adventitious root. Oppositely, IAAOL2 expressed during all the stages, but the expression level has the negative relationship with rooting ability. Which means we can not determine its functions of the rooting process of Larix.(6)Simulation experiments showed that IAAOL1 and IAAOL2 expressed higher in treatment with 1×10-3M NAA. After being treated with different hormone, IAAOL1 and IAAOL2 expressed highest in material which was treated with auxin. After being treated with different auxin, IAAOL1 and IAAOL2 expressed highest in material which was treated with NAA. After being treated with NAA, IAAOL1 and IAAOL2 expressed highest in material which was treated after 24 hour. All of results showed that IAAOL1 and IAAOL2 are auxin early response genes. IAAOL1 and IAAOL2 may improve the growth of adventitious root in Larix cutting.(7)During the different stages of the rooting process of Larix cutting, GA20OL expressed higher in the species which have high rooting ability. gibberellin promotes the growth of adventitious root of Larix cutting by opposing the effects of nuclear DELLA protein growth repressors.In a word, the formation of adventitious root of Larix cutting was very complex process, the genes related to hormones played pivotal roles, as the same time, the other genes were very important too. Some novel function-unknown genes on formation of adventitious root of Larix cutting were found. 34.2% genes obtained could not be found significant homologous sequences(E-valus>0.001) in GenBank. The results indicated the formation of adventitious root of Larix cutting was not known clearly in gene level. Additionally, function-unknown genes obtained from Larix could be studied further in the model plants by comparing to genome of A. thaliana. In conclusion, our study maked it well suited for dissection of rooting in Larix cutting and it is a milestone to carry out the molecular study of rooting in Larix cutting.