Identification And Function Analysis Of GhWRKY15,a Novel WRKY Gene From Cotton (Gossypium Hirsutum L.)

As sessile organisms, plants lack circulating, somatically adaptive immune systems. Inthe long evolutionary process, plants develope some specific immune systems for adaption tovarious environments. Sometimes, these environments may restrict plants growth anddevelopment. In this specific immune systems, many regulatory genes function via beinginduced or repressed, and they either directly protect plants from attacks or further alterexpression of other related genes. Thus, plant defence responses are regulated by a complexnetwork of signalling pathways which fine-tune expression patterns of defence-related genes.Among these complicated signalling pathways, the transcriptional activation ofdefence-related genes requires expression of particular transcription factors or alterations oftheir DNA-binding activity.WRKY transcription factors，as a superfamily of regulatory proteins, play importantroles in adapting to various environments and in the process of plant growth and development.Although lots of studies have revealed the role of WRKYs in these aspects, the mechanismsunderlying the ways that WRKY members act their role still need to be further investigated.Moreover, previous researches mainly focus on the WRKY members in Arabidopsis and rice,but studies focusing on the WRKY transcription factors in cotton crops are rather limited. Inthis thesis, we firstly identified a IId WRKY gene, and explored part of its function viaanalysis of amino acid sequence, subcelluar location, expression patterns and diseaseresistance. The main results are as follows.（1） A putative WRKY family member gene, nominated GhWRKY15, was identified fromcotton. An amino acid sequence analysis showed that the deduced GhWRKY15proteinshared42.60%,49.70%,54.27%and63.19%similarity to NtWRKY3（BAA77358）,AtWRKY15（NP₁79913）, StWRKY2（ABU49721） and PtWRKY16（ACV92028）,respectively. GhWRKY15presented as a single copy gene. DNA structure analysis showed that GhWRKY15has two conserved introns. In addition, subcelluar location analysis indicatedthat GhWRKY15was located in the nucleus.（2） Analysis of the part GhWRKY15promoter sequence revealed a number of cis-actingelements related to the response to environmental stresses and regulation of plant growth anddevelopment process.（3） Compared with wild-type tobacco, GhWRKY15-overexpressing tobacco showedmore resistance to both viral and fungal infections. Furthermore, several pathogen-relatedgenes, NPR1and two genes encoding enzymes involved in ET biosynthesis showed increasedtranscripts accumulation in the GhWRKY15-overexpressing tobacco under disease attacks.（4） Importantly, increased activity of the antioxidant enzymes POD and APX underdisease infection and enhanced expression of NtAPX1and NtGPX in transgenic tobacco undermethyl viologen treatment were detected. Northern blot analysis indicated that expressionlevel of GhWRKY15was significantly upregulated in cotton seedlings under fungal infectionor salicylic acid, methyl jasmonate or methyl viologen treatment.（5） GhWRKY15exhibited higher transcription level in the roots and stems compared within the cotyledon of cotton seedlings, which may explain the phynotype that the stems oftransgenic plants displayed earlier shooting stages compared with wide type tobacco.（6） In addition, GhWRKY15expression was enhanced in cotton seedlings when exposedto GA3, ABA and several abiotic stresses, including cold, wounding and drought.In conclusion, our results suggest that GhWRKY15, as a regulator of the ROS system,acts its role in the response to both viral and fungal infections via multiple signallingpathways. It is intriguing that GhWRKY15is involved in plant growth and development,especially in stem development. These findings imply that the role of the WRKY proteins indisease resistance may be closely associated to their regulatory role in plant growth anddevelopment.