Functional Analysis Of Tomato LeNLP4Transcription Factor

In the last decade, NAC(NAM, ATAF1/2, CUC2) transcription factors which are unique to plants have been the new type transcription regulatory factors. And all NAC proteins constitute one of the largest families of plant specific transcription factors. Members of NAC transcription factor family play important roles in transcription activation and inhibition, including formation of apical meristem and secondary cell wall, development of lateral roots and embryo, ripening of plants and plant responses to biotic and abiotic environmental stresses. Consequently, study on LeNLP4is important to root growth and stress resistance of NAC transcriptional factors regulating plant defense to temperature stress.We cloned a NAC transcription factor which named LeNLP4from the tomato leaves in this study. Overexpression of LeNLP4promoted root growth, enhanced chilling tolerance of transgenic tomato plants and heat stress tolerance of transgenic tobacco plants. The main results are as follows:(1) We isolated the full length cDNA of LeNLP4by RT-PCR. The full length of LeNLP4is1595bp and the ORF is1218bp encoding406amino residues. LeNLP4has a conserved NAC domain in the N-terminal and the C-terminal acting as transcription activating domain. Transcripts of LeNLP4accumulated to the highest level in roots. The expression of LeNLP4was higher in flowers and friuts and was induce by chilling, heat, salt, drought, and hormones.(2) LeNLP4::GFP fusion protein was transiently expressed in onion epidermal cells and we found that it was located in the nucleus by using confocal laser scanning microscope.(3) Prokaryotic expression vector pET-LeNLP4was constructed and transformed into E.Coli BL21. We used IPTG to induce proteins, which was purified and used to immunize the mice, and then we could obtain antiserum. The titer of antibody was1:100,000. The results of Western blot showed that the expression levels of LeNLP4protein were increased in over-expression lines.(4) The full-length ORF of LeNLP4was subcloned into the expression vector pBI121under the control of the35S-CaMV promoter to form sense and antisense constructs. The Agrobacterium tumefaciens-mediated leaf disk method was used to generate transgenic tomato plants. Kanamycin-resistant plants were detected by PCR, qRT-PCR and Western blot. The results suggested that over-expression lines and antisense seeding which influenced formation of apical meristem and died were obtained. Compared with wild-type plants (WT), the roots of and the dry weight of over-expression lines increasd.(5) Compared with WT, transgenic tomato plants have higher growth increment and maximal photochemistry efficiency of photosystem II (PSII, Fv/Fm), higher capability for scavenging hydrogen peroxide (H2O2) and superoxide radical (O2-), higher scorbate peroxidase (APX) and superoxide dismutase (SOD), and lower malondialdehyde (MDA) content and the relative electric conductivity (REC) under chilling treatment (4℃). Expression level of CBF1in transgenic lines was higher than that in WT. These results indicated that overexpression of LeNLP4enhanced chilling tolerance of transgenic tomato plants.(6) Compared with WT, transgenic tobacco plants have higher Fv/Fm, higher capability for scavenging H2O2and O2-, higher activities of peroxidase (POD) and SOD, and lower MDA content and REC under heat stress (42℃). These results indicated that overexpression of LeNLP4transcription factor enhanced heat stress of transgenic tobacco plants.