Isolation and homogenization of hrpW and hrpG genes for transfer to citrus in order to create resistance to citrus canker

Abstract

Canker disease is caused by bacteria (Xanthomonas citri pv. citri), which every year reduces the yield and quality of citrus family products, especially limes and oranges. This research was conducted with the aim of isolating and homogenizing two genes encoding harpins HrpW and HrpG from Xcc bacteria strain NIGEB-088 and making recombinant plasmids pBI-hrpG and pBI-hrpW in order to develop a resistance strategy to canker disease based on induction of defense reaction in lime plant. For this purpose, specific primers (back and forth) were designed for the amplification of these genes using the complete genomic DNA sequence of the bacterium causing this disease. The amplified genes were then inserted into pGEM7zf(-) and pUC19 plasmids for hrpW and hrpG genes, respectively, with XbaI and SacI, as well as XbaI and BamHI cutting sites, and the recombinant plasmids were transformed into E.coli strain DH5?. In order to express the genes in the plant, the desired fragment was homogenized in the pBI121 vector and in the cutting sites for the target genes, so that the desired genes were under the control of the 35S cauliflower mosaic virus promoter and the NOS terminator in the T-DNA region of this vector. Using polymerase chain reaction, enzymatic digestion and sequencing, the presence of target genes in positive colonies was confirmed. These recombinant plasmid vectors can be used in gene transfer to plants sensitive to this disease, such as lemons and oranges, using Agrobacterium. Finally, the recombinant plasmids were transferred to the desired strains of Agrobacterium tumefaciens and the presence of the relevant genes was confirmed. Approved strains of Agrobacterium tumefaciens will be used in gene transfer programs.           

Key words: citrus canker, gene homology, Xanthomonas citri bacteria, harpin genes

1-Introduction

1-1-Introduction to citrus

Compounds with the scientific name of Citrus spp. They are from Rutaceae family and Auranutideae subfamily. Citrus are bushy, shrubby plants with dense foliage, most citrus species (such as orange[1], grapefruit[2] and tangerine hybrids[3]) in subtropical regions with cold winters flower only once a year in late winter and early spring. But in tropical and coastal areas, citrus trees such as lemons[4] and limes, which flower in spring and summer, may flower several times throughout the year or have a very long flowering period (Davis et al., 1994). In citrus, the flowers have 4-8 thick white, red or purple petals, 4-5 sepals and 16-32 stamens. The ovary has 6-14 elliptic carpels attached to a very narrow, sometimes swollen and wide stigma that ends in a spherical stigma. Flowers in citrus fruits are bisexual, and also the type of pollination of citrus fruit is different according to the species and they are self-fertile, self-fertilizer-transformed and parthenocarpous. For example, limes are generally self-indulgent (Castell and Gemiter, 1999).

Citrus fruits are rich in vitamins A, B, C, fiber, carbohydrates (simple sugars, fructose, glucose, and sucrose) and amounts of calcium, potassium, niacin, and folic acid, which lower blood cholesterol, prevent viral infections, and reduce the risk of colon cancer. and stomach (Gornstein et al., 2001). Citrus production is very important in the world today and it is one of the important sources of wealth, commercial exchanges and employment for the residents of around 137 citrus-rich countries in the world. About a hundred industries in the world use citrus fruits in the production of their products. One of the important and significant things in the citrus industry is the high added value of this product through the production of its by-products, which include pharmaceutical, food and cosmetic raw materials (Ismail and Zhang, 2004).

Citrus cultivation takes place between the latitudes of 40 degrees north and south of the equator, which is at the commercial levels of the main citrus production areas of the world between the latitudes. 5/23-40 north and south degrees are located. The minimum temperature required to start citrus growth (physiological zero) is 12.5 degrees Celsius and the average suitable temperature for optimal growth is 18.5 degrees Celsius.The minimum temperature required to start the growth of citrus fruits (physiological zero) is 12.5 degrees Celsius and the average suitable temperature for optimal growth is 18.5 degrees Celsius. Citrus fruits need 180 hours of heat collection per month (Fatohi et al., 2015). Temperature and water significantly influence the time and period of flowering, flower distribution, percentage of fruit formation, and the state of growth and fruit fall of citrus trees. Citrus fruits need 750 mm (15,000 cubic meters of water per hectare) of water per year in wet areas and 1500 mm (15,000 cubic meters of water per hectare) per year (Khoshkhoi et al., 2013). Cold and frost are among the environmental issues that pose a risk to citrus fruits [5] and pests [6].

The best type of soil for citrus cultivation is sandy-loamy soil, and they also perform well in the pH range of 5.5 to 5.7. Among horticultural crops, citrus fruits are the most sensitive group to soil salinity (Khoshkhoi et al., 1364).

1-2-History of citrus tree cultivation in Iran and the world

The origin of citrus fruits can be found in Southeast Asia, China, and the Indian Archipelago and from 2400 years before Christ (Jamitar and Ho, 1990). The southern shores of the Mazandaran Sea are also mentioned as the second focus of citrus cultivation (Elahinia, 2013). The introduction of citrus fruits to Iran, except for Balang, has a history of 400 years. According to historical documents, Iran has been the gateway to the exit of citrus fruits from Asia to other parts of the world (Elahinia, 2013).

1-3- Cultivated area, production rate and performance of citrus trees in Iran and the world

According to the citrus statistics report of the Ministry of Agriculture in 2013, the cultivated area of ??citrus trees in the three central, northern and southern regions of the country is 288,108 hectares and the amount of production was 4,299,247 tons, which indicates the population's willingness to consume and, as a result, the necessity of more importance to the research and cultivation of this product in order to meet the food needs of the growing population of the country. The efficiency[7] of blue citrus production in the country is 16931.6 kg per hectare. Mazandaran and Lorestan provinces have the highest and lowest water yields with 21832.2 and 438.7 kg, respectively.

According to FAO statistics in 2008, the area under citrus cultivation in the world is 8.7 million hectares and the average production of citrus fruits in the world is reported to be 122 million tons. The survey of the major citrus producing countries shows that China, Brazil, Nigeria, Mexico, America, India, Spain and Iran occupy the first to eighth place in terms of cultivated area. Meanwhile, based on the amount of citrus fruit production, the countries of Brazil, America, China, Mexico, Spain, India, Italy and Iran are ranked first to eighth respectively (Amar Nameh Kashwarzi, 2018). In terms of performance[8], citrus fruits are ranked first to fifth, respectively, by America, Turkey, South Africa, Japan, and Argentina, while Iran ranks tenth in this respect.

Despite the existence of the necessary potential for citrus cultivation in the country, the existence of limiting factors and also the lack of proper management have caused Iran to have only 0.4% of the world trade[9] of citrus fruits (product statistics). Baghi, 1387), which indicates the need to conduct more studies on these biological [10] and non-biological [11] limiting factors and manage these factors using modern knowledge and technologies in order to reduce their impact on the cultivation, production and export of this product. 1-4- Importance and types of citrus diseases rtl;"> The importance of plant diseases for humans is due to the damages [12] that are caused to plants and their products as a result of the disease. Plant diseases may be the limiting factor for planting a plant in a region or a country and destroy all the plants of a species that are sensitive to a particular disease (Ashkan, 2015). Despite the differences in terms of the amount and type of financial losses caused by diseases, an informed and knowledgeable farmer can use the best available variety, agricultural, biological, genetic and Chemical, not only to have proper management for his crop, but also to get more profit even in the years when other orchards suffered a lot of losses.