| As an agricultural state, crop’s yield, rural incomes and food security are important base of economic and social progress in China. Because of growing population and dwindling arable lands, the contradiction between population and land resource surges increasingly. Those contradictions provide new challenges and opportunities for cotton industry. Currently, our country has large-area dry and saline-alkali lands need to be explored. Breeding new transgenic cotton variety with high salt and drought tolerance by molecular design breeding technology, it is not only important for utilization of dry and saline/alkaline lands and cotton industry development, but also for our country’s economic and social progress.In general, plants exhibit slow growth, premature and low yield under conditions of drought and salt. With analysis of plants growing on stressful conditions, it suggested that plans respond to abiotic stresses through multiple pathways, such as increasing osmotic adjustment substances and controlling cell cycle to increase cell division and growth. It is reported that glycinebetaine is a kind of ideal non toxic osmotic regulator, which regulate osmotic pressure of plant cell, maintain the structure of biological macromolecular and protect the function of photosynthetic system. In plants, glycinebetaine(GB) is synthesized by two-step, which need choline monooxyegenase(CMO) and NAD-dependent betaine aldehyde dehydrogenase(BADH). NEK6 encodes a serine-threonine kinase, and it involves in regulating cell cycle related gene expression and ethylene signal transduction. In this study, we enhance tobacco and cotton tolerance in salt and drought stresses through two strategies, including improving accumulation of GB and increasing At NEK6 expression. Those results will provide the foundation for breeding cotton varieties with drought-resistant and salt-tolerant. Results from our experiment are summarized as follows:1. Ah CMO and Ah BADH were cloned into vector p BI121 to establish Ah CMO-Ah BADH two-gene vector and Ah CMO/Ah BADH single-gene vector for transformation of tobacco. In the text, OE-CB represents transgenic line of transforming by Ah CMO-Ah BADH two-gene vector; OE-C and OE-B represent transgenic lines of transforming by Ah CMO and Ah BADH single-gene vector, respectively. T2 transgenic plants were obtained. Those genes were expressed normally in transgenic plant leaves. Compared with wild type(WT), the contents of GB were increased in transgenic plants, and the contents of GB in OE-CB were 2.1 times as higher as OE-C and OE-B, and 10.9 times as higher as WT. The MS medium contained 200 m M mannitor or 150 m M Na Cl were used for drought- or salt-tolerant test. The results showed that root length and lateral root number of transgenic plants were better than WT. Root length of OE-CB was 24.2% and 26.5% longer than OE-C and OE-B, respectively. Lateral root number of OE-CB was 47.2% and 58.5% higher than OE-C and OE-B, respectively. In greenhouse experiments, the transgenic plants grow better than WT, and fresh weight of OE-CB was increased 33.8% and 34.7% more than that of OE-C and OE-B, respectively.Based on the analysis of osmotic pressure test, drought and salt stress can increase osmotic pressure of plant cells. Osmotic pressure increased multiple of transgenic plants were higher than those of WT, and the osmotic pressure of OE-CB were 56.3% and 51.3% higher than OE-C and OE-B. The results of chlorophy Ⅱ fluorescence parameters showed that physiological characteristics of photosynthesis in transgenic plants were superior to WT. OE-CB lines were higher than OE-C and OE-B lines. Therefore, the growth of transgenic plants under drought and salt stress environment were related to osmotic pressure and plant physiological characteristics of photosynthesis. The activity of Superoxide Dismutase and Catalase showed that transgenic plants have better abilities to degrade hazardous substances such as hydrogen peroxide and superoxide under stressful conditions. The results of malondialdehyde content determination showed that MDA contents of OE-CB lines were the lowest, OE-C and OE-B lines took second place, and WT was the highest, indicating that the degree of membrane and lipid peroxidation of OE-CB were lowest. In addition, the content of proline was increasd by transgenosis of Ah CMO and Ah BADH. Therefore, the transformation with Ah CMO and Ah BADH minimize the damage to the plants caused by adverse conditions.In brief, with analysis of the transgenic lines’ s phenotype, including root length, lateral root number, growth vigour and fresh weight, cell osmotic pressure, and the activity of SOD and CAT and so on, we concluded that all three types of transgenic lines displayed better drought and salt tolerance than WT, and OE-CB line displayed best of the three. From this point of view, we transformed cotton by Ah CMO-Ah BADH two-gene vector to select high drought- and salt-resistant transgenic cotton. Currently, we have gotten 20 transgenic cotton plants.2. At NEK6 was transformed into tobacco and obtained T2 transgenic progenies. At NEK6 can express as expected in transgenic plants by q RT-PCR test. Under normal condition, root length, lateral roots number and fresh weight of transgenic plants was increased 6.1%, 6.3% and 5.9% more than that of WT, respectively. In drought and salt treatments, transgenic plants grew better than WT. Root length, lateral roots number and fresh weight of transgenic plants were 2.0, 1.7, 2.3 times as higher as WT under drought treatment, and those results were 2.1, 2.0, 2.3 times as higher as WT under salt treatment. The results of Fv/Fm showed that physiologyical characteristics of photosynthesis of transgenic plants were superior to WT. Under drought and salt stress treatment, the activity of SOD and CAT showed that transgenic plants have better abilities to mop up reactive oxygen compounds than WT. The Analysis results of MDA showed that the degree of membrane and lipid peroxidation of transgenic plants were lower than WT. The contents of proline in transgenic plants were higher than WT.The results above indicated that the biomass of transgenic plants were increased under non-stress condition, the salt- and drought-resistant of transgenic plants were increased under stress condition by introducing At NEK6 gene. The At NEK6 gene can be used to breed salt-tolerant and drought-tolerant cotton varieties. In this study, we transformed At NEK6 into cotton and obtained transgenic embryonic callus.In conclusion, we conducted a systematic study of genes involving in two different abiotic stress pathways in tobacco, and our results confirmed that the transformation with Ah CMO, Ah BADH and At NEK6 genes significantly improved the ability of tobacco resistance to drought and salt stresses. In addition, we established those genes vector, which involving in two pathways, and obtained cotton transgenic progenies. These results will provide the basis for using two kinds stresses pathways related genes to improve cotton resistance to drought and salt. |
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