Overexpressing Citrate Synthase And Phytase Gene In Brassica Napus Improved Tolerances To Phosphorus Deficiency And Aluminum Toxicity In Soil

Aluminum (Al) toxicity and low phosphorus (P) availability are two major constraints on crops growth in acid soil. Although P availability is very low, total P is quite abundant in soils with organic P constituting up to20%-80%, which is the potential P source for plant growth. Brassica napus is one of the main oil crops and is cultivated up to more than80%of cultivated region of oil crops in China. However, these cultivated region of Brassica napus are acid or relatively acid soil, with high Al toxic levels and low P availability. So, reducing or eliminating Al toxicity of acid soils and improving soil P availability are essential to enhance Brassica napus production.In this research, based on the Agrobacterium-mediated gene transformation, we introduced a Pseudomonas aeruginosa citrate synthase (CS) gene and two phytase genes phyA and appA which include an extracellular targeting sequence from the carrot extension (ex) gen into Brassica napus cv Westar10. The tolerances of the transgenic lines to Al toxicity and P deficiency, and their physiological and molecular mechanism were studied using hydroponics, sand and soil culturing in the study. The main results were listed as follows:1. Five T3generation transgenic Brassica napus lines were obtained by molecular identification and Km-resistance screening. The results of Southern blotting showed that CS3and CS6transgenic lines overexpressing CS gene harboured two and single P. aeruginosa CS loci, respectively. P3and P11transgenic lines harboured single and three A. niger phyA loci, respectively, and a18transgenic line harboured two E. coli appA loci. Northern blotting analysis showed that transgenic lines CS3and CS6showed an accumulation of P. aeruginosa CS transcripts. The expression of A. niger phyA at the mRNA level was very high in P3and P11transgenic lines. a18transgenic lines showed an accumulation of E. coli appA transcripts. All of the five transgenic lines are lack of segregation with kanamycin-resistant screening.2. Transgenic lines CS3and CS6overexpressing CS gene improved the tolerance to Al toxicity. Both root concentration and exudations of citrate and malate in transgenic lines CS3and CS6significantly increased compared with wild type (WT) following exposure to Al. These may be attributed to higher activities of the CS, malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) enzymes in the TCA cycle and the expression of BnALMT and BnMATE in the transgenic plants following Al exposure.Transgenic lines CS3and CS6have enhanced Al tolerance. When exposed to25μM AICI3for48h, the relative root lengths of transgenic plants are significantly longer than WT. Moreover, prolonged Al treatment (10days) experiments revealed that transgenic lines accumulated much more biomass than WT.3. Transgenic lines CS3and CS6overexpressing CS gene improved the tolerance to P deficiency. Transgenic lines CS3and CS6showed enhanced citrate and malate exudation when grown in P-deficient conditions. Moreover, the enzyme activities of the transgenic lines were significantly higher compared with WT in response to P-deficient stress. The soil culture experiment showed that transgenic lines CS3and CS6possessed improved P uptake from the soil and accumulated more P in their shoots and seeds when FePO4was used as the sole P source.4. Transgenic lines P3, P11and a18overexpressing phytase gene enhanced the tolerance to P deficiency. The extracellular phytase activities of transgenic Brassica napus overexpressing ex::phylappA are significantly higher than WT in hydroponic culture. Quartz sand culture experiment showed that the shoots P accumulation in the three transgenic lines P3, P11and a18increased by70.8%,36.8%and19.6%compared with WT, respectively, when phytate was used as the sole P source. Moreover, the shoots biomass of the three transgenic lines was all significantly higher than WT. The soil culture experiment showed that seed yields of transgenic lines P11and a18increased by20.9%and59.9%, and seeds P accumulation increased by20.6%and46.9%compared with WT, respectively, when phytate was used as the sole P source. Phytase activities in P3, P11and a18seeds reached approximately1000units per kg seed, whereas no phytase activities were detected in WT seeds. Moreover, phytic acid contents of P11and a18seeds were significantly lower than WT.In conclusion, the overexpression of the CS gene in B. napus can not only lead to increased citrate synthesis and exudation, but also altered malate metabolism. The increased rate of the two organic acids exudation in transgenic canola overexpressing CS lead to significantly improved capacities for Al tolerance and soil P acquisition. The increased extracellular phytase activities in transgenic canola overexpressing ex::phylappA lead to significantly improved the ability of plants to utilize phytate. Moreover, transgenic seeds of P3, P11and a18have highly phytase activities.