Transcriptome analysis, biochemical characterization and tissue culture regeneration of leucaena leucocephala

Abstract

Leucaena leucocephala (leucaena) is a leguminous tree, which is adapted well to tropical or subtropical semi-arid environments, highly resistant to both biotic and abiotic stresses, and a protein-rich forage for livestock animals. The long-term goal of this research is to identify leucaena genes for defense to plant pathogens, insects-pests and tolerance to environmental stresses, including drought and salinity. The specific objectives of this project were: (i) transcriptome analysis of genes expressing in shoots and roots of leucaena, (ii) biochemical characterization of transgenic leucaena expressing a bacterial dioxygenase-hydrolyase fusion gene (pydA/pydB) to reduce mimosine content of its foliage, and (iii) tissue culture regeneration and multiplication of transgenic leucaena plants. To explore the molecular basis of leucaena's drought tolerance, insect and pathogen resistance, and mimosine biosynthesis, total RNA was extracted from the shoots and roots of three-month-old plants and sent to SeqWright Inc. for transcriptome sequencing. A total of 1,022,583 and 1,165,136 scaffolds were obtained from the transcriptome sequences of shoots and roots of leucaena, respectively. The numbers of contigs obtained were 1,047,350 (in shoots) and 1,190,291 (in roots). All the transcriptome analyses of leucaena in this project were based on 199,818 (in shoots) and 112,091 (in roots) scaffolds, which showed similarities with gene sequences in the NCBI database. Among these, 35,177 and 4,745 scaffolds, that were larger than 500 bp, were used for further analysis of the shoot and root sequences. A total of 33 root sequences were identified that were absent in shoots. Several classes of potential genes for resistance to both biotic and abiotic stresses were identified in the transcriptome sequences. A total of 74 and 160 chitinase sequences were found in the root and shoot transcriptomes, respectively. As expected, a large number of disease resistance genes, encoded by NB-LRR type of genes, were found in the leucaena transcriptome. In the shoot transcriptome, the number of NB-LRR sequences (>500 bp) was 86; among these 36 NB-LRR sequences were >1.0 kb. In contrast, only 18 NB-LRR sequences (>500 bp) were identified in the root transcriptome. A large number of WRKY transcription factors, some of which may be involved in disease resistance, were also identified in the root and shoot transcriptomes; their numbers were 145 and 223 for roots and shoots, respectively. Similarly, 109 sequences encoding different members of ERD (early responses to dehydration) family of genes were identified. These genes may be involved in drought resistance. For UV tolerance, 11 and 3 gene sequences (>500 bp) were found in the shoot and root transcriptomes, respectively. A total of 636 sequences (>500 bp) encoding different types of Ser/Thr kinases, including 29 sequences showing high similarities to receptor Ser/Thr kinase, were identified. Furthermore, 33 sequences (>500 bp) encoding histidine kinases, 22 sequences (>500 bp) encoding different types of tyrosine kinases as well as receptor tyrosine kinases, 21 sequences (>500 bp) encoding various types of PTPs, 18 MAPK sequences were identified. Thirty sequences (>100 bp), 23 from roots and 7 from shoots, encoding cysteine/mimosine synthase were also identified. In spite of having many desirable attributes, leucaena contains a toxic non-protein amino acid mimosine, which is harmful to animals. Our laboratory is developing transgenic leucaena plants expressing a bacterial pydA/pydB fusion gene under the control of a CaMV 35S promoter. We expected that these plants should contain reduced amounts of mimosine. I had determined mimosine contents of sixteen transgenic plants. All transgenic leucaena plants showed lower mimosine content compared to the wild type leucaena. The transgenic leucaena plants contained 40.47-95.9% less mimosine than the wild type. Among these, three plants (# 1, 3, 5) were found to contain only small amounts of mimosine (0.06-0.10% of dry weight). Currently, there is no suitable method for tissue culture micropropagation of leucaena. To multiply the transgenic leucaena plants, I improved the protocol of tissue culture. Using shoot tips and nodal segments, four plants were regenerated from the transgenic plant #3. Identification of many genes for diseases and drought resistance from leucaena through transcriptome analysis has opened new areas of research for the future. The disease and drought resistance genes can be characterized further by isolating full-length cDNA. These genes can be used for developing disease and drought resistant varieties of other crop plants. Biochemical determination of transgenic leucaena plants has established that 3 among 16 transgenic lines contain only small amounts of mimosine. These plants will be valuable as a fodder for farm animals. The micropropagation method for leucaena developed in this research will be helpful for rapid multiplication of transgenic leucaena plants without having to wait for the plants to flower and produce seeds.