Cloning And Functional Analysis Of EβF Synthase Genes

Aphids are major agricultural pests, partly due to their serious physical and economic damage to cultivated plants by sucking nutrients from the phloem or by transmitting plant viruses. However, the plant defence against aphid-resistance among the available wheat germplasm was seldom identified with the mechanism underlying remains unclear. Chemical insecticides provide a simple strategy for wheat aphid control. However, the large-scale application of such chemicals is not in an environmentally friendly way and becoming increasingly unacceptable. Transgenic wheat engineered for enhanced aphids′resistance could be an efficient alternative strategy. Some plant lectins, including Galanthus nivalis agglutinin (gna) and Pinellia ternate agglutinin (pta), have shown to be toxic towards aphids in transgenic wheat. However, it is reported that gna transgenic plants could cause the sterility of predatory ladybird via aphids in its food chain, and biosafety issues related to the application of these genes need to be evaluated. Therefore, other safe and effective genes for aphid control need to be exploited. EβF [(E)-β-farnesene], as the main and sometimes only component of most aphid alarm pheromone, could cause other aphids in the vicinity to become agitated or disperse from their host plant. What is more, EβF can also function as a kairomone to attract the predator of aphids.Here, the EβF synthase genes were isolated from peppermint,sweet wormwood (Artemisia annua) and douglas fir (Pseudotsuga menziesii), respectively. Functional analysis of individual EβF synthase gene was carried out with transgenic tobacco plants obtained. Moreover, one of the isolated EβF synthase genes, MhβFS1, was transferred into wheat for the first time. The results are indicated as following:1. Cloning and functional analysis of the EβF synthase genes from peppermint: (1) MhβFS genes were isolated from Mentha haplocalyx Briq and Mentha x piperita by RT-PCR. Two EβF synthase genes, named MhβFS1 and MhβFS2, were discovered in Mentha haplocalyx Briq. Compared with MhβFS1, there was a substitution of Val to Ala at position 361 for MhβFS2. The sequence of EβF synthase gene from Mentha x piperita was the same as that of MhβFS1. The length of MhβFS1 and MhβFS2 genomic sequence were 2690 and 2753 bp, respectively. Both of them contain six introns with the same intron phase (intron phase of the six introns is 0, 1, 2, 2, 0 and 0, respectively). (2) qRT-PCR confirmed that MhβFS could express in all the three selected organs, the expression level in leaves was somewhat higher than that of the roots and stems. (3) The molecular weight of heterologous recombinant MhβFS1/MhβFS2 was about 63 kD. The recombinant MhβFS1 was purified using HisTrapTM HP before injecting into a rabbit. The titer of the rabbit′s anti-serum was higher than 125 000. (4) Twelve MhβFS1 positive transgenic individuals were identified by PCR, some of them were further analyzed by southern blot, qRT-PCR and western blot. GC-MS showed that transgenic lines emitted EβF at a level of 243 ng per day for Mh1-1, 398 ng per day for Mh1-2, 644 ng per day for Mh1-3 and 769 ng per day for Mh1-4, respectively. Compared with the control plant W38, transgenic lines showed repellence to aphids with the number of aphids on Mh1-1, Mh1-2, Mh1-4, Mh1-6 decreased 7.0%,9. 3%,16.3% and 14.0%, respectively.2. Cloning and functional analysis of the EβF synthase genes from sweet wormwood: (1) Two EβF synthase genes, designed as AaβFS1 and AaβFS2, were isolated from Artemisia annua. The deduced protein of AaβFS1 had four amino acids differing from that of AaβFS2 including a Leu deletion at position 21, a Asp to Asn at position 50, a Leu to Ile at position 89, a Gly to Arg at position 509. The cDNA sequences of AaβFS1 and AaβFS2 were deposited in GenBank under accession number GU294840 and GU294841, respectively. The length of AaβFS1 genomic sequence was 2392 bp, containing six introns with the intron phase of 0, 1, 2, 2, 0 and 0, respectively. (2) Eleven AaβFS1 and five AaβFS2 positive transgenic tobacco plants were identified by PCR. Southern blot, qRT-PCR and SDS-PAGE further confirmed the integration, expression of the transgenes in transgenic lines. According to the results of GC-MS, AaβFS1 and AaβFS2 transgenic tobacco plants could emit EβF at a level of 217~706 ng/day. The number of aphids on transgenic line Aa1-4-5 decreased 10.0% compared with the non-transgenic control W38. Interestingly, transgenic tobacco plants showed special attraction to green lacewings (Chrysoperla carnea). Then four hundred alate aphids and ten lacewings were simultaneously introduced into a setup, twelve hours later, the number of aphids was reduced by approximately 16.4% in transgenic line Aa1-3-4, 23.6% in Aa1-4-5, 10.9% in Aa2-1-2, 20.0% in Aa2-3-6, respectively. Therefore, we would like to presume that, AaβFS1 and AaβFS2 lines could have a pleiotropic effect on aphid behaviors, especially attraction to aphid predators for aphid control.3. Cloning and functional analysis of the EβF synthase genes from Douglas fir: (1) Two different PmβFS cDNA clones, designed as PmβFS1 and PmβFS2, were isolated from Douglas fir. Both the ORF of PmβFS1 and PmβFS2 contained 2478 nucleotide, which encoded 825 amino acids. The deduced protein of PmβFS1 had four amino acids differing from that of PmβFS2 with 98.9% identities. The length of PmβFS2 genomic sequence was 3846 bp, containing eleven introns. (2) The molecular weight of heterologous recombinant PmβFS1/PmβFS2 protein was around 63 kD, existing in the inclusion body. (3) Fifteen PmβFS1 and five PmβFS2 positive transgenic tobacco plants were identified by PCR and qRT-PCR. GC-MS indicated that transgenic tobacco emitted small amounts of EβF, perhaps due to the activity of PmβFS1 and PmβFS2 synthesas were low in transgenic tobacco plants.4. Preliminary researches on MhβFS1 transgenic wheat: The expression vectors both for particle bombardment, i.e. MhβFS1-pG4AB and MhβFS1+CTP-pG4AB, and Agrobacterium-mediated transformation vectors such as MhβFS1-pGПUB and MhβFS1+CTP-pGПUB were constructed with rice rbcS promotor to direct the expression of target genes with chloroplast transit peptide, ?-kozak sequence added to the 5'-end of MhβFS1 and poly(A) sequence to the 3'-end. MhβFS1 was then transformed into wheat cultivar Yangmai 12 and Kenong199 by biolistic and Agrobacterium-mediated methods, respectively, Positive transgenic wheat lines were advanced to T3 generation. Southern blot of some T0 transgenic wheat lines indicated that transgenes were integrated into the genome of transgenic wheat. Further analysis of EβF emission of the transgenic lines and its effect on the bioassay for aphid control is now under way.