A NOVEL APPROACH TOWARDS THE MOLECULAR IDENTIFICATION, BIOINFORMATICS ANALYSIS AND ROLE OF EIN3 AND EIL1 GENES FAMILY IN COTTON FIBRE
BY; Tahir Iqbal
Background and Problem Statement
Ethylene has a significant role in the growth of plants such as maintaining the elongation processes as well as development of cotton fiber (Cao et al., 2017). One of the most important parameter in elongation of cotton fiber is the biosynthesis of ethylene. Production of ethylene in appropriate amount significantly increases the fiber development in Gossypium hirsutism. Inositol synthase and expansion are involved in fiber development in cotton. Ethylene overall controls the gene expression of inositol synthase and expansion.
Fatty acids that are long chain in nature also involved in elongation of cotton fiber (Doglike et al., 2019). EIN3 and EIL1 (Ethylene Insensitive3/Ethylene Insensitive 3) are the main transcriptional factors that are specifically playing an important role in signal transduction in cotton. Both these factors are involved in regulating the signaling responses induced by ethylene. EIN3 and EIL1 are mainly involved in regulating the transcription by activating the other factors that involved in transcription such as ethylene-response-factor (Hu et al., 2017; Zhang et al., 2018). EIN3 and EIL1 also regulate the signal transduction in Arabidopsis by regulating the expression of GCC box.
EIN3 binding F-box proteins have a significant role in regulating the responses induced by ethylene in Arabidopsis (Hu et al., 2017). The structure of EIN3/EIL1 comprises mainly two biochemical features in relation to sequence of amino acids that maintain the functional activities of these proteins (Riau et al., 2003). Firstly, EIN3/EIL1 proteins contain special sequences of special amino acids at the amino-terminal that are highly conserved.
Secondly, the sequences of the carboxy terminal of EIN3/EIL1 proteins are lower and less conserved as compared to the sequences of the amino terminal (Song et al., 2015). There are several functions of EIN3/EIL1genes for regulation of biochemical pathways in some plants such as Arabidopsis and tomato (O’Malley et al., 2016). Molecular mechanism of transcription is initiated by responses of ethylene and regulation of transcription is crucial in Arabidopsis (Harley et al., 2018). Biochemical roles as well as cellular properties of genes associated with EIN3 and EIL1 have been studied in various plants such as rice, tobacco, tomato and Arabidopsis (Li et al., 2019).
EIN3 gene play vital role in the regulation of transcription and also acts as a main transcriptional regulator in response to Sulphur metabolism in Arabidopsis (Maruyama Makushita et al., 2006). EIN3/EIL1genes show a relationship with ethylene as well as Sulphur metabolism in plants. Nicotiana tabacum EIL2 is engaged in the regulation of expression of some genes in tomato by binding with the UP9C promoter. (Shi et al., 2018).
The role of EIN3 and EIL1genes in the development of cotton fiber has not completely described. Molecular regulation of EIN3 and EIL1genes at transcriptional level in cotton remains unclear. The molecular mechanism and biochemical process of the development of cotton fiber in which EIN3 and EIL1genes play a main role remains unclear.
Fiber mutants are the main useful source for describing the molecular mechanism in the development of cotton fiber. Transformation of gene in the crop plants is one of the landmark achievements of biotechnology. Genetic transformation in crops plant can be achieved by number of methods like Agrobacterium Ti-plasmid vectors, micro projectile bombardment, and micro-injection, PEG treatment of protoplast and electroporation of protoplasts.
All these procedures have certain impacts over each other but transformation via micro projectile bombardment and Agrobacterium are mostly used now days and will be preferred during this study (Fang et al. 2018).
Objectives
The objectives of this study will be categorized into following key points
To examine the gene expression analyses of EIN3/EIL1 genes involved in development of cotton fiber through RT-q PCR.
To perform the Phylogenetic Analysis by determining the sequences of amino acids of EIN3/EIL1 genes.
To observe the functional information about EIN3/EIL1 genes through conducting the Bioinformatics analysis
To check the interaction among EIN3/EIL1 genes through constructing the specific PPI networks of EIN3/EIL1.
Materials and Methods
1. Study Plan
This study will be probably conducted under School of Agricultural Sciences, Zhengzhou University/Institute of Cotton Research of CAAS.
Sample preparation, specific standard solutions and buffer solutions will be prepared according to the instructions given by instructor. Laboratory instruments will be used according to following SOPs. Bioinformatics analysis will be performed according to instructions given by supervisor. This study will be carried out into following sections.
1.1. Plant Materials
Plant materials such as upland cotton seeds would be collected from Chinese Academy of Agricultural Sciences, China, Research institute and grownup under natural condition. Cotton flowers will be labeled and bagged properly to protect from self-pollination.
All cotton samples will be collected by following the instructions given by supervisor. The collected samples will be immediately frozen under liquid nitrogen.
1.2. Blotting technique
To find out a specific gene, single piece of DNA among thousands is the key to know further about genetic study. For this purpose, blotting technique is a powerful tool and mechanically quite simple process to extract out desired actions (Krane, 2002). In this experiment, Southern blotting technique will be followed to extract DNA of Gossypium hirsutism and to carry out further processes and bioinformatics analysis.
1.3. Description of EIN3/EIL1 genes
The genomic data of Gossypium hirsutism would be taken from cotton gene database. To perform BLAST p search, protein sequences of EIN3/EIL1 of Arabidopsis will be utilized as queries and these protein sequences will be taken from TAIR database. For identification of EIN3/EIL1 genes in Gossypium genome finely, in NCBI; in Conserved Domain Database (CDD) EIN3/EIL1 domain will be searched for protein sequences (Li et al., 2019). Bioinformatics tool such as BLAST p will be used for identification of transcription factors of EIN3/EIL1genes. After complete description of these, EIN3/EIL1 genes will be named manually.
1.4.Location on chromosome
Map Chart software will be used to find out the location of EIN3/EIL1 genes on chromosomes. Expanse software will be used to examine the molecular mass, size and biochemical parameter such as isoelectric point of EIN3/EIL1 genes. Wolf PSORT software will be used to predict the subcellular positions of EIN3/EIL1 genes. SMART software will be used to check the main domain of EIN3/EIL1 genes (Pawer kiwis et al., 2017).
1.5 Molecular Analysis of EIN3/EIL1 genes
Samples analysis will be carried out for the RT-q PCR experimental investigation by using the Applied Biosystems-7500 RT-PCR detection systems (Applied Bio systems, USA). Specific primers will be designed for EIN3/EIL1 genes that will be used for experiments conducting through RT-q PCR (Zhang et al., 2015). Three replicates will be taken for every sample during the experimental investigation. Expression analysis of the EIN3/EIL1 genes involved in development of cotton fiber will be carried out through RT-q PCR. Further, STRINGTIE will be used for measuring the different levels of expression.
1.6.Phylogenetic Analysis
Sequences of amino acids of EIN3/EIL1 genes will be aligned by using the Cluster W method. Phylogenetic relationships will be analyzed by using the MEGA 6 software and neighbor joining method will be used with Poisson correction as well as replicate of 1000 bootstraps (Thyssen et al., 2014).
1.7. GO-Gene Ontology of EIN3/EIL1 genes
Gene Ontology will be performed to access the functional information about EIN3/EIL1 genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the EIN3/EIL1 genes will be performed by classification and characterization of genes at functional level with using the principles of functional enrichment analysis. Functional enrichment software that will be used for classification and characterization of genes at functional level (Pathan et al., 2017).
1.8.Protein-Protein Network (PPI) Analysis
The interactions among proteins using bioinformatics software helpful for better understanding the functions of specific genes found in plant. Biological database such as STRING will be designed for to construct the specific PPI networks of EIN3/EIL1 due to known PPIs and analysis will be performed to check interactions among proteins (Szklarczyk et al., 2015).
1.9.Gene Expression Analysis
Biological database such as Plant CARE will be used to analyze the cis-elements in specific sequences of promoters of EIN3/EIL1 that are especially involved in transcription of genes. Ps RNA Target will used to analyze the gene expression of mRNA target of the G.hirsutum EIN3/EIL1(Dai et al., 2018).
1.10. Gene transformation
5-enolpyruvylshikimate-3-phosphate (EPSPS). A glyphosate resistant EIN3/EIL1 resilient genes from CP4 strain of Agrobacterium will be subjected into the cotton cultivar involving the techniques of Agrobacterium mediated transformation of shoot-apex.Primary transform ants will be partitioned using 0.26% spray of glyphosate and successively PCR analyses. Different transformed will be obtained from T1 seed and transgenic insertion (incorporation of the EIN3 and EIL1) will be analyzed by southern-blotting technique. Transcription of gene and protein expression level in transgenic cotton cultivar will further examined by real time-PCR, western blotting and ELIZA techniques cotton genome presented the uptake, incorporation and expression of the EIN3 and EIL1will be analyzed in cotton cultivar (Fang et al. 2018).
Research benefits
This research will be helpful in elucidating the role of EIN3 and EIL1 genes for better understanding the molecular mechanism for the development of cotton fiber in future studies. This research based study will also be helpful in explaining the interactions of EIN3 and EIL1genes with different molecular pathways that are involved for better development of cotton fiber.
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