Cloning And Functional Identification Of WRKY From Corylus Heterophylla Fisch

In agricultural production, low temperature (frozen) cold harm is a kind of serious naturaldisasters, which give rise to gigantic loss every year in the world. Therefore, plant hardinessmechanism study not only plays an important role in the production in theory, but also have thewide application value in production. Corylus heterophylla Fisch. is the peculiar germplasmresources in China, which can tolerate low temperature-48℃, however, the research related toits transcription factors of cold resistance has been rarely reported. WRKY transcription factorsas an important transcription factor family responding to the abiotic stress, plays a fundamentalrole in plant hardiness. In this study, we focused on the members of WRKY transcriptionfactors screened from transcription library of Corylus heterophylla Fisch. winter bud, andrelated genes. Meanwhile, we analyzed its expression under natural conditions and artificialadversity stress, respectively. Finally, we obtained the key transcription factors of WRKYrelated to cold resistance from Corylus heterophylla Fisch., which possess an significantmeaning for the future breeding of new varieties resistant to cold by molecular assistedbreeding method and transgenic breeding engineering. The main results are as follows:1. Getting the Unigene sequence of49WRKY transcription factors, totally, in whichthere were26sequence of Unigene fragments that can be campared to poplar, indicating thatWRKY sequence of transcription library from Corylus heterophylla Fisch. has an quite closerelation with the WRKY transcription factor family of public database in poplar as woodyplants. Designing the3'RACE and5' RACE primers according to the target WRKY Unigenesequence, we cloned and obtained14WRKY transcription factors, named ChWRKY1,ChWRKY2, ChWRKY28, ChWRKY7, ChWRKY13, ChWRKY10, ChWRKY11, ChWRKY18,ChWRKY32, ChWRKY9, ChWRKY33, ChWRKY20, ChWRKY4, ChWRKY6respectively. Thereare5genes of the14WRKY transcription factors from Corylus heterophylla Fisch. belong tothe Ⅰt ype of WRKY,8genes belong to the Ⅱ type, especially theChWRKY6belongs to theⅢ type in WRKY family, moreover, zinc finger structure of which is the C2-HC.2. We took the ChActin as internal parameter and made a preliminary study for the expression of7ChWRKY genes in bud during the winter. The results indicate that under naturalconditions,ChWRKY7, ChWRKY6, ChWRKY13in November has the highest expression,ChWRKY1, ChWRKY2, ChWRKY28, ChWRKY20is in December.Then, ChWRKY1, ChWRKY2,ChWRKY28, ChWRKY7expression began to decrease gradually with the season change,ChWRKY20, ChWRKY6, ChWRKY13in March has a slightly increase instead.3. Expression of ChWRKY genes from Corylus heterophylla Fisch. were analyzed underartificial4℃low temperature, salt, drought condition, respectively. The results showed thatthe expression of ChWRKY2, ChWRKY28, ChWRKY7, ChWRKY6, ChWRKY13were allincreased induced by4℃low temperature, salt and drought stress, what's more, the responseto low temperature and drought treatment is relative earlier, and later to salt stress. In addition,ChWRKY1expression rise induced by the low temperature stress, while ChWRKY20inducedby salt and drought stress, lesser induced by the former stress.4. Nucleus location analysis of WRKY transcription factor from Corylus heterophyllaFisch. showed that after infecting cells, p35S-WRKY28-GFP restructured plasmid only hadgreen fluorescent signals in the nucleus,while in both cytoplasm and nucleus in the control,which indicating WRKY28is an Nucleus location protein that is consistent to forecastingresults through the software. In other words, WRKY28as transcription factors, play part in thenucleus.5. Build plant expression vector PBI121-WRKY28, will build a good expression vectorby agrobacterium mediated, pollen tube channel method into arabidopsis, won the WRKY28transgenic arabidopsis strain. Will the wild type and genetically modified arabidopsis4℃processing, found that wild type arabidopsis at4℃processing after12h wilting phenomena arevery obvious, and genetically modified arabidopsis wilting is relatively small.6. Obtained the two genes ChMAPKKK and ChMAPKK of MAP kinase cascade systemfrom Corylus heterophylla Fisch. through the RACE technology.The overall length ofChMAPKKK was2396bp, including one1704bp open reading frame, coding568amino acids,and the molecular weight was61.68kD, and theory isoelectric point (pI) was4.75. The overalllength of ChMAPKK was1665bp, including one1062bp open reading box, coding354aminoacids, and the molecular weight was39.32kD, and theory isoelectric point (pI) was5.82. The typical phosphoric acid consistent sequence of ChMAPKK was the SLADIDS. In our study wealso found that the general expression trends of ChMAPKKK and ChMAPKK was basicallyconsistent with WRKY of Corylus heterophylla Fisch. under drought,salt,and low temperaturestress.7. The soluble protein, SOD and POD enzyme activity, MDA, proline content in leaf ofCorylus heterophylla Fisch. under low temperature, drought and salt stress were analysesed,which had different variation trend under different adeversity treatment. Analysing the relativeelectric conductivity, soluble protein content, SOD, CAT, POD enzyme activity and the contentof MDA in leaf of the wild type and transgenetic arabidopsis after12h by low temperaturetreatment, the results showed that the relative electrical conductivity, the increase of the contentof MDA in transgenetic arabidopsis were less than wild type.The more the content of MDA,the more serious the degree of Lipid peroxidation in membrane. The incrase in soluble proteincontent, SOD enzyme activity in transgenetic arabidopsis were higher than wild typearabidopsis thaliana after12hours of treatment. The different changes of biochemical indicatorhappened in transgenetic arabidopsis so that to improve the resistance to low temperaturestress.