Identification Of Genes Related To Root Development In Citrus Rootstock And Its Functional Study

Drought is one of the important restricting factors to the development of citrus industry.As the underground part of fruit trees,root system of rootstock is the most important organ for plants to absorb water and nutrients directly from the soil.Therefore,to a certain extent,the ability of the rootstock to absorb water determines the drought resistance of the plant.Different rootstocks have different ability to absorb water depending on their genetic background.Studies in several crops have found that root morphology and drought resistance are closely related.Certain parameters of the root system morphology have been used as the main selection parameters for drought resistance breeding,and related genes that control root morphology and structure have been identified in model plants such as Arabidopsis,rice,and corn.Drought resistance can be affected by regulating the expression of those related genes.However,the current research on drought resistance in citrus mainly focused on the morphological charactors and physiological state of the leaves,and research on root system are very rare.Therefore,in this study,citrus rootstocks with different genetic backgrounds were used as test materials to analyze and identify the morphological characteristics of citrus roots.At the same time,transcriptomic analysis was carried out to deeply excavate and identify the genes related to root morphological development.The main results are as follows:（1）Comprehensive evaluation of root systems of different citrus rootstocksIn this study,10 kinds of citrus rootstocks with different genetic backgrounds were used as experimental materials,including:Hongningmeng（Citrus limonia Osbeck cv“Hongningmeng”）,XC-009（C.junos）,Z-006（Poncirus hybrid）,Carrizo citrange（C.sinensis×Poncirus trifoliata cv Carrizo citrnage）,Ziyangxiangcheng（C.junos cv“Ziyangxiangcheng）,Shiikuwasha（C.depressa Hayata cv“Shiikuwasha”）,Z-021（Poncirus trifoliata）,KPJ-030（C.reticulata）,ZZ-030（Poncirus hybrid）,ZZ-022（Poncirus hybrid）,WEKM（C.volkameriana Pasquale cv‘Volkamer’）.The main root length,total root length,total root surface area,total root volume,average root diameter,and number of root tips were determined at 30,45 and 75 days after sowing.The results showed that the root system morphology and structure of 11 citrus rootstocks were significantly different.The results of comprehensive evaluation indicated that the values of WEKM,Z-021 and ZZ-022 were higher than those of Z-006,Ziyangxiangcheng,XC-009,ZZ-030 and KPJ-030,while those of Carrizo orange,Hongningmeng and Shiikuwasha were the lowest.The 11 rootstocks can be divided into three groups by cluster analysis based on the parameters of morphology as:Better root system（WEKM,Z-021）,medium root system（Z-006,ZZ-030,KPJ-030,XC-009,ZZ-020,Ziyangxiangcheng）,poor root system（Carrizo orange,Hongningmeng,Shiikuwasha）.The results of cluster analysis are consistent with the comprehensive evaluation results.（2）Identification of genes related to root development by transcriptomic analysisAccording to the root phenotype,the long root type of Volkamer,Canton lemon,Z-023 and short root type of Shiikuwasha,Z-11and Z-007 were used as the experimental materials to construct the RNA pools of long root type and short root type.The transcriptome sequencing and bioinformatics analysis were carried out to screen the candidate genes related to root development for further verification.The results showed that 41.66Gb Clean Data was obtained in this transcriptome sequencing,and the Q₃₀ base percentage of each sample was not less than 89.68%,which indicates that the sequencing quality is qualified and can be used for subsequent analysis.Results of gene expression analysis showed that a total of 661 differentially expressed genes were obtained,including 429 up-regulated genes and 232 down-regulated genes.10 candidate genes were found to be related to root development of the rootstock and were selected for q RT-PCR analysis.The expression of Ciclev10032781m.g（ABA receptor protein PYL4 gene）in three short-root rootstocks of Shiikuwasha,Z-11and Z-007 was significantly lower than that of the long-root rootstock.This was consistent with the results of transcriptomic analysis.This candidate gene should be related to the root development of the rootstock.Therefore,it was taken as the subject of subsequent research.（3）Functional identification of citrus Cc PYL4 geneNine Cc PYL gene family members were identified from the whole genome of C.clementina.The theoretical isoelectric points of the Cc PYL proteins are ranged from 4.9 to 8.88,and the number of amino acids encoded are 168 to 245.The results of subcellular localization prediction showed that the members of Cc PYL gene were located in different positions of the cell,most of them in the nucleus or chloroplast,and a few of them in the cytoplasm and cell membrane.Analysis of the original homeopathic promoters showed that the upstream sequences of Cc PYL genes contain the elements for light response,hormone response,tissue specificity,biological processes and abiotic stress response.q RT-PCR analysis results showed that most of Cc PYL genes could respond to exogenous hormone and abiotic stress treatment.After ABA treatment,the expression of Cc PYL4showed a trend of first decrease and then increase.The Cc PYL4 was cloned from WEKM and Shiikuwasha.The similarity between the sequences of those two Cc PYL4 was very high（99.8%）.There was only one nucleic acid difference.It is a synonymous mutation.Tissue-specific expression analysis showed that the expression of Cc PYL4in roots,stems,and leaves was significantly different.The expression level in roots was much higher than that of in stems and leaves.The PGBI-Cc PYL4 overexpression vector was constructed and transformed to Arabidopsis thaliana.Five 5 positive plants of T₃ generation were obtained.q RT-PCR results showed that the expressions of Cc PYL4 in five transgenic plants were 2500-5500 times than that of the control.The results of root morphology evaluation showed that the lengthes of main root of the transgenic plants were 1.78-2.27 times than that of the control.The differences were significant.The above results indicate that Cc PYL4 participates in the regulation of citrus root growth and development,and can promote the elongation of main root.