Identification And Expression Pattern Analysis Of Phytocystatin Genes In Mulberry Plants

Agricultural pests are the main reason that leads to crop losses. People always use chemical pesticides and traditional breeding of resistant varieties to control pests. However, the excessive use of chemicals enhances the insecticide resistance constantly and these chemicals seriously pollute soil and water. Breeding system has many advantages such as economy, security, and so on. But it is limited due to the long period and unstable resistance. Much attention has been paid to obtain efficient and stable insect-resistant materials using the plant transgenic technology. Thus, identifying the genes that can effectively resist insects is necessary and a critical step. Since proteinase inhibitors can inhibit the digestive proteinase of insects and then improve the nutritional quality of food, the genes encoding these inhibitors are regard as important targets for pest control.Phytocystatins belong to cysteine protease inhibitors, and they are widely distributed in plant, animal, and microorganism. Because most of coleoptera insects use cysteine proteases to digest food, phytocystatins interfere with the coleoptera insects’digestive system and have no specific inhibition to lepidoptera insects. So it is important to identify phytocystatin genes in mulberry and thus be used in pest (mulberry longicorn) control strategies using transgenic technology.Mulberry is a kind of plants that can secrete latex. It has been reported that a great variety of proteins and secondary metabolites in latex participated in the process against insects feeding. Except silkworm, Bombyx mori, many insects in nature can not feed on mulberry leaves. Identification of proteinase inhibitors in mulberry latex will provide new targets for us to develop stable and efficient nsecticidal trangentic systems.In this research, we identified the phytocystatin genes in the mulberry genome using bioinformatic methods. Meanwhile, qRT-PCR was used to study the transcriptional levels of the identified genes in different tissues of M. notabilis and Morus atropurpurea. The expression of the identified genes under MeJA, wounding, and feeding treatment were aslo detected. Transient transformations of onion epidermal cells were performed and was used to study the subcellular localization of MaCPI-1. Western-blotting was carried out to study the protein expression patterns of MaCPI-1 under different treatments. The main results are as follows:1、Identification and analyses of mulberry phytocystatin genesWe identified six putative phytocystatin genes in the M. notabilis genome database by bioinformatic methods. These genes were named as MnCPI-1, MnCPI-2, MnCPI-3, MnCPI-4, MnCPI-5, and MnCPI-6. All of the identified genes were cloned in M. notabilis and Morus atropurpurea with only several different bases. Gene structure analysis revealed the number and position of introns were generally conserved in these genes. Conserved motif analysis showed that six phytocystatins contatin three motifs that were involved in the interaction with their target proteinases. The phylogenetic tree which included 51 deduced amino acid sequences of phytocystatins identified in mulberry and other plants were constructed by neighbor joining method. The tree was classifed into three major groups (A, B and C), the proteins encoded by the six phytocystatin genes in mulberry were distributed in three groups. Interestingly, MnCPI-1, MnCPI-2 and MnCPI-3 were distributed in group A and Phytocystatins, which had C-terminal extension were all grouped into this group. MnCPI-6 was clustered into group B with phytocystatins of monocotyledonous plants. MnCPI-4 and MnCPI-5 were distributed in group C, whose phytocystatins were all derived from dicotyledonous plants. The genes with introns were clustered in group A and intronless genes were clustered in groups B and C. The results indicated that gene structure was consistent with that of phylogenetic tree.A total of 17 deduced amino acid sequences of phytocystatins that have C-terminal extension were used to construct phylogenetic tree. The result indicated that the mulberry MnCPI-1 was closely clustered to apple and strawberry, both of which also belonged to the Rosaceae family. They shared 75% and 70% identical amino acid residues to MnCPI-1, respectively. And the high identity of amino acids within motifs suggested that they might have the conserved biological functions. This C-terminal extension region of MnCPI-1 showed similarity with cystatins from other plant species. Genes, which have C-terminal extension, in a total of 10 species, include mulberry, do not detect positive selection in the overall evolution by the site-model analysis, suggesting that mulberry MnCPI-1 gene may be neutral or purifying selection.The three-dimensional structure prediction exhibited that MnCPI-3, MnCPI-4 and MnCPI-5 shared very similar configuration to OC-I reported previously. However, specific features were observed for some of the mulberry phytocystatins, for instance, MnCPI-2 lacked of theβ1-sheet at the N-terminus, and two minor loops were predicted in the middle of β1- and β2- sheets of MnCPI-6.The cis-acting regulatory elements in promoter regions of mulberry phytocystatin genes were analyzed in plantCARE database. Three types of cis-elements were included as light-responsive elements, hormone-responsive elements and stress-responsive elements, which suggested that they might be involved in protecting plants against attack by pests, fungi, drought and cold resistance.2、Expression analysis of phytocystatin genes in mulberryqRT-PCR method was used to study the transcriptional levels of the identified genes in roots, stems, flowers, leaves and latex from M. notabilis and roots, stems, leaves from Morus atropurpurea, respectively. The results indicated that 6 phytocystatin genes were expressed in at least one of the five tissues,4 phytocystatin genes(MnCPI-1～MnCPI-4) were expersssed in all tested tissues with various levels. MnCPl-5 showed tissue-specific expression in flower, and MnCPI-6 had much lower expressions in roots, stems, flowers and latex, while it’s hard to detect in leaves. At the same time, except MaCPI-5, the 5 phytocystatin genes were expressed in all tested tissues of M. atropurpurea, with differential expression levels.Only the expression pattern of MaCPI-3 was consistent with that in M. notabilis. It indicated that the tissue expression pattern of phytocystatin genes were differential between M. notabilis and M. atropurpurea.Compared to other plant origins, mulberry phytocystatin genes were widely expressed in different tissues. In additionto the individual genes, most of genes can be detected in roots, stems, flowers, leaves and latex. It suggested that the phytocystatin genes might play diverse biological roles in mulberry.qRT-PCR was used to study the transcriptional levels of phytocystatin genes under MeJA, wounding and feeding treatment in Morus atropurpurea leaves. Except for MaCPI-5, other five genes were up-regulated in different degrees under the treatments, and the up-regulation of genes is more obvious under the MeJA treatment. The expression patterns of different genes were basically identical under the same treatment, and so were the expression patterns of the same gene under different treatments.3、Subcellular localization and protein experssion analysis ofMaCPI-1The analysis of subcellular localization of MaCPI-1 was performed by means of agrobacterium-mediated transformation of onion epidermal cells. And, the data showed that it was distributed in the nucleus and cytoplasm. Western-blotting method was applied to study the induced protein expressions of MaCPI-1 by MeJA, wounding and insect feeding in Morus atropurpurea. The results showed that MaCPI-1 was expressed in normal physiological conditions and were upregulated by above treatments, suggesting the putative role for inhibiting insect digestive proteinase.In this study, six phytocystatin genes in the mulberry genome were identified and cloned. Bioinformatic analyses indicated that they shared several futures with other plant cystatins comprising sequence identity, gene structures, conserved motifs, and three-dimensional structures. qRT-PCR analyses showed that mulberry phytocystatin genes were widely expressed in tested tissues with various levels. In addition, these genes responded to MeJA, wounding and insect feeding treatments. Subcellular localization and western-blotting experiments indicated that MaCPI-1 was a secretory protein, protein exoression of which was increased in response to above treatments. It was suggested that MaCPI-1 might be involved in inhibiting the insect digestive proteinases. Present study, for the first time, reported the identification of mulberry phytocystatin genes, and selected significant candicate genes using molecular biological technique, which provided basic data for future genetic improvement.