Cloning And Phylogenetic Analysis Of Laccases In Maize And Ryegrass And Gene Expression Of Water Stress In Maize

Maize is the second largest crop for food and feed in China. Forage quality and yield of maize are greatly affected by digestibility and drought stress. Conventional maize breeding has played an important role in improving forage quality and drought tolerance, but it is time-consuming and labor intensive. The rapid progress in molecular biology and genomics has provided an opportunity in accelerating forage improvement and dissecting mechanism of drought tolerance.In the present study, the laccase genes in ryegrass and maize were cloned and compared, while the gene expression of four maize inbred lines under water stress were analyzed, based on gene homology sequence cloning, cDNA-AFLP and bioinformation techniques. This work may provide theoretical and practical guides for accelerating and facilitating breeding for good forage quality and drought tolerance in maize and thus contribute to the structural adjustment of agricultural production and sustainable agriculture in China. The main results are as follows:1. A comprehensive phylogenetic analysis of 64 laccase genes from monocots, dicots and gymnosperm was conducted to identify putative orthologues and paralogues within the multiple-gene family. By multisequence alignments, a wide range of conserved domains and motifs were found in most members. Phylogenetic analysis indicated that plant laccase genes are a highly divergent multigene family. Duplication of laccase genes has been a continuous process occurring before and after the seed plants diverged into gymnosperms, monocots, and dicots.2. Plant laccases from different plant species, and in some cases, even within the same species, had a relatively low level of sequence homology. Both extracelluar and intracellular plant laccases were found.The range of pI values of plant laccase, substrate specificity, and numbers of N-glycosylation site varied greatly. Furthermore, plant laccase genes were confirmed to be of tissue-specific expression, inferring that this enzyme group is likely to carry out diverse functions in the plant.3. A total of 27 and 17 laccase genes from two model plants, rice and Arabidopsis. respectively, were used to construct frame maps. Of the 27 laccase genes from rice, nine were located on chromosome 1 and 6 on chromosome 11, indicatingthat more than half of the members concentrated on two chromosomes. Laccase genes mapped on rice chromosome 1 were dispersed into three regions that spanned 21Mb estimated from GRLC26 to GRLC1. GRLC1 was located around region 1 (15371897-15374072 bp) whereas GRLC6 was located around region 2 (25214631- 25216628 bp). Region 3 ranged from GRLC5 to GRLC1 (1.3 Mb), which spanned between C126435 and C15960 according to the published data on chromosome 1. On chromosome 11, the laccase genes also were divided into three regions. Four of them were located at the distal region of short arm. Comparing with the published map of chromosome 11 based on a doubled haploid population, it was found that GRLC11,12 and 15 were close to a gene cloned from rice, designated as a MYB transcription factor. Of the 17 Arabidopsis laccase genes, only AtLCl and AtLC7 were dispersed on chromosomes 1 and 4,...