Expression And Interaction Analysis Of BlKNOX Genes In Betula Luminifera

KNOX homeodomain transcriptional factors have been shown to play an important role in plantsdevelopment.KNOX proteins can be further classified into two subgroups on the basis of sequencesimilarity.KNOXI genes are expressed in shoot apical meristems, and they determine the cell fate ofshoot apical meristems and control the initiation and development of the lateral organs;KNOXII geneshave some diverse expression patterns and few known of functions.KNOX II genes have been reportedto regulate the formation of secondary cell wall,xylem,root and other organs.In plants,KNOX proteinsregulate hormones in the meristem homeostasis by regulateing its target genes.They also interact withother homeotic domain subfamily BELL proteins and then regulate gibberellin and lignin biosyntheticpathways.In this study, isolation, expression and interaction analysis of KNOX genes in Betulaluminifera.We gain several main results below.1.Five KNOX genes,named as BlKNOX1-BlKNOX5,were cloned from Betula luminifera by usinghomologous cloning and RACE methods.KNOX domain were analyzed through Blast,the results showthat the five genes belong to typical KNOX gene families,its coding protein containsKNOXA,KNOXB,ELK,HD domain structure.Sequence alignment of BlKNOX1-BlKNOX5to a numberof KNOX genes showed that BlKNOX1,3,4belonged to KNOXI genes while BlKNOX2,5are highlyhomologous to the KNOX genes of Class II.At the same time,Real-time PCR Expression analysis forBlKNOX1-BlKNOX5genes were performed.The results showed that BlKNOX2,5main expression in theleaf,and with the development of leaf,the expression showed the tendency of lowered after the first andamount reaches the maximum value in the immature leaf. BlKNOX1,3,4main expression in the stem,and as the degree of lignification, BlKNOX1showed a trend of gradually reduce the amount of itsexpression,while BlKNOX3,4showed a trend of gradually rise the amount of its expression.2. BlKNOXs were cloned into vector pGBKT7to generate bait plasmids, Then we transformedpGBKT7-BlKNOXs into yeast strain Y2HGold to test the self-activation and toxicity. The resultsshowed that pGBKT7-BlKNOX2,4,5had no autonomous activation and could be used in the yeast two-hybrid system,while the others had self-activation effect.In order to understand BlKNOX1,3,s the keydomain structure of the activation effect of key domain structure,different truncated forms ofBlKNOX1,3were constructed bait plasmids, and transfected into yeast Y2HGold cells,and theirautonomous activation and toxicity were examined. The results showed that four truncated forms ofBlKNOX1were not have utonomous activation and toxicity,it can infer that the integrity of the geneticstructure of whether BlKNOX1bait plasmid have utonomous activation. N-terminal protein ofBlKNOX3in yeast Y2HGold had toxicity, the KNOXB of BlKNOX3may be the key domain structureof its utonomous activation,the existence of the N-terminal and KNOXA domain may be enhance theactivity of utonomous activation.3.The yeast two-hybrid system library has been constructed. According to the growth of colony counting,7.5×107library containing transformants.The insert size by PCR from the colony and plasmidsfrom the library was from0.4to3Kb,indicating that the library database meets the requirements and laythe foundation for yeast two hybrid test.4. BlBELs were cloned into vector pGADT7to generate prey plasmids and tested the interaction ofBlBELs and BlKNOXs in yeast.The results showed that besides BlBEL1and BlKNOX2withoutinteraction, the rest were detected interact, but there are differences between the strong and weak. At thesame time expression analysis were performed and showed that BlKNOX2,5and BlBEL6,7with thedevelopment of leaf,the expression showed the tendency of gradually rise.The expression ofBlKNOX1,2,5and BlBEL2,6,7in the development of female flowers showed the tendency of up-regulation.It could infer that both BlKNOX2,5and BlBEL6,7together to participate in the regulation ofleaf development.BlKNOX1,2,5and BlBEL2,6,7co-regulated in the development process of the femaleflowers.5.pGBKT7-BlKNOX2bait plasmid was selected to screen Betula luminifera yeast two-hybridlibrary,AbA,X-α-Gal and SD nutrient medium were used to identify the positibve clones.Plasmids DNAwere isolated from223positive clones and used to transform E.coli to prepare plasmids,which weresequenced by ABI genetics analyzer.After sequence comparison and Blast analysis against the NCBIdatabase,twenty five positive clones from the screening were found which encode transcriptionalregulators,peptidases,phosphatase,lysozymes.These results lay the foundation on the reveal of thefunction and mechanism of Betula l. KNOX genes. At the same time expression analysis of BlKNOX2and the sequences of screened Betula l. cDNA library expression analysis were performed and showedthat BlKNOX2and transcription factor of Myb family had the tendency of gradually rise in leaf.BlKNOX2and proteins which regulated flowering presented the tendency of gradually rise in femaleflowers.It could infer that Infer BlKNOX2may play a role in leaf and female flowers developmentprocess.