摘要
水稻是全世界最重要的粮食作物之一,占全球谷类作物种植面积的1/3,世界上约有50%的人口以稻米为主食。在作物所有必需营养元素中,氮是限制植物生长和形成产量的首要因素。氮(N)素是作物从土壤中吸收量最多的元素,其对作物的生命活动和产量形成具有重要意义。土壤中植物所利用的主要氮素形式是铵态氮(NH4+)和硝态氮(NO3-)。尽管在淹水条件下土壤中氮素主要以铵态氮形式存在,但是有研究表明水稻根系能够泌氧,从而在硝化细菌作用下将根际土壤中铵态氮转化为硝态氮,在潮湿土壤状况下水稻根系吸收硝态氮占总氮量的25%～40%。植物为了能够适应土壤中N03-浓度梯度差异环境,进化出N03高、低亲和吸收转运系统。近年来人们从分子水平上对硝态氮的吸收系统进行了大量研究。已发现两类基因NRT1和NRT2分别于与低亲和硝态氮转运系统(LATS)和高亲和硝态氮转运系统(HATS)有关。目前已从多种植物中克隆了高、低亲和硝态氮运输系统的基因,特别是对模式植物拟南芥的研究较为深入。
尽管在水稻中已经克隆到了五个NRT2家族的硝酸盐转运蛋白基因,但是对它们功能特性的研究还十分有限。本试验以水稻高亲和硝酸盐转运蛋白基因OsNRT2.3a和OsNRT2.3b为研究对象,利用超表达及RNAi技术获得OsNRT2.3a/b超表达和OsNRT2.3a RNAi转基因材料；通过RT-PCR.qRT-PCR、TAIL-PCR.Southern blot、 Western blot等方法分析转基因材料超表达及沉默效果；15N同位素示踪分析转基因材料对硝酸盐的吸收及转运；田间试验分析转基因材料的产量性状以及利用基因芯片对转基因材料全基因组表达进行分析,研究了水稻OsNRT2.3a/b基因的生物学功能,所获得的主要研究结果如下：
1.通过生物信息学分析表明水稻高亲和硝酸盐转运蛋白基因OsNRT2.3在转录过程中通过选择性剪切产生OsNRT2.3a和OsNRT2.3b两个转录本,OsNRT2.3a和OsNRT2.3b在cDNA序列上差异非常小,OsNRT2.3b相对OsNRT2.3a有一个90bp长的内含子,且5’端和3’端非编码区都长于OsNRT2.3a;芯片结果表明田间生长条件下OsNRT2.3a/b在根部表达量最高,地上部叶片也有表达,其他部位表达量较低；从完整生育期来看,OsNRT2.3a/b在叶片中的表达量随着植株的生长而增高,在植株营养生长向生殖生长过渡的阶段达到最大值,之后表达量降低到一个相对稳定的范围；水稻原生质体亚细胞定位实验表明OsNRT2.3a和OsNRT2.3b都是细胞质膜定位的跨膜蛋白,与生物信息学预测结果一致。
2.通过水稻转基因分别得到了OsNRT2.3a、OsNRT2.3b的超表达转基因水稻材料和OsNRT2.3a RNAi沉默转基因水稻材料,通过RT-PCR鉴定了目的基因超表达及RNAi的阳性株系,并且通过southern blot和TAIL-PCR筛选到了独立的单拷贝插入在基因组非编码区的转基因株系,进一步通过Western blot验证了蛋白水平上的超表达及沉默效果,通过繁种获得了遗传性状稳定的超表达及沉默转基因株系。
3.通过OsNRT2.3a和OsNRT2.3b的超表达转基因水稻植株的表型发现单独超表达OsNRT2.3a基因的转基因植株与野生型水稻相比没有明显的表型差异,而单独超表达OsNRT2.3b基因的转基因植株与野生型水稻相比株型显著增大。对超表达植株中OsNRT2s和OsNAR2.1的表达分析表明,OsNRT2.3a超表达并没有影响OsNRT2.1、 OsNRT2.2和OsNAR2.1的表达；OsNRT2.3b超表达植株中OsNRT2.1. OsNRT2.3a和OsNAR2.1的表达都显著上调,而OsNRT2.4的表达量基本不受影响.在水稻中单独超表达OsNRT2.3a基因并不能明显影响水稻的生长,也没有提高水稻对氮素的吸收；在不同浓度及形态的氮素处理条件下,OsNRT2.3b超表达植株生物量都显著大于野生型植株,而总氮浓度除了在高浓度铵态氮处理下与野生型无显著差异外,其他条件下均较野生型低,但整个植株的总氮含量在OsNRT2.3b超表达植株中都要显著高于野生型。
4.对OsNRT2.3b超表达植株与野生型在不同施氮水平下的田间试验表明,OsNRT2.3b超表达水稻长势在各个施氮水平下均要好于野生型,且平均单株产量和氮素利用效率都显著高于野生型。同时我们也发现,OsNRT2.3b超表达水稻的生育期相比野生型有所推迟,其差异主要体现在抽穗期比野生型推迟,且随着施氮量的增加,OsNRT2.3b超表达水稻生育期推迟的天数也随着增加。进一步对OsNRT2.3b超表达植株生育期差异的研究表明,OsNRT2.3b超表达不仅能提高水稻干物质积累总量和后期干物质转运量,而且能显著提高水稻后期干物质转运率和转运干物质贡献率；OsNRT2.3b超表达不仅能提高水稻氮素积累总量和后期氮素转运量,而且能显著提高水稻后期氮素转运率和转运氮贡献率。
5.通过对OsNRT2.3a RNAi沉默突变体的分析表明,OsNRT2.3a基因沉默不会显著影响外界低NO3-条件下水稻根系对NO3-的吸收,同时OsNRT2.3a也没有参与硝酸盐从老叶(氮源)到新叶(氮库)的再移动,但OsNRT2.3a基因的沉默会抑制N03-从地下部往地上部的转运,从而导致水稻根系NO3-的积累,进而间接下调OsNRT2.1/2.2的表达量,最终导致水稻根系减少从外界环境中吸收硝酸盐。
6.对OsNRT2.3a超表达及沉默突变体的全基因组表达谱芯片分析表明OsNRT2.3a沉默突变体由于地上部氮素含量不足影响植株碳氮循环,进而影响到植株的光合作用,导致干物质积累减少、植株生长矮小；OsNRT2.3b超表达植株全基因组表达谱芯片分析表明OsNRT2.3b基因超表达可能适当抑制了光呼吸的同时提高了植株体内抗氧化活性,从而延缓植株的衰老,提高植株干物质的积累及产量。
综上所述,水稻高亲和硝酸盐转运蛋白基因OsNRT2.3a主要在硝酸盐从地下往地上部的运输过程中起关键作用；OsNRT2.3b超表达能够显著提高水稻前期对氮素的积累和后期对氮素的转运,进而提高水稻氮素利用效率和增加产量。
Rice(Oryza. sativa L.) is one of the most important staple food crops that provides food for more than half of the world's population, accounting for1/3of the global cereal crop acreage. Nitrogen is one of the major factors limiting crop growth and yield among the necessary nutrient elements for plant. Both ammonium and nitrate are the major sources of soil N for plants, however, ammonium is the predominant form of N in a paddy soils. Some physiological experiments have shown that lowland rice was exceptionally efficient at acquiring NO3-formed by nitrification in the rhizosphere, and nitrate was main source of N in soil at later growth and development stages of rice under the intermittent irrigation. It has been predicted that25%～40%of the total N taken up by rice roots grown under wetland conditions might be in the form of nitrate and the rate of nitrate uptake can be comparable with that of ammonium. To meet the different NO3-concentration in the soil, higher plants have low-affinity nitrate transport system (LATS) and high-affinity nitrate transport system (HATS) to uptake nitrate. In higher plants, the molecular basis of root nitrate uptake has been the matter of intensive studies during the last decade. So far, two gene families have been identified:the NRT1and NRT2families involved in the low-affinity nitrate transport system (LATS) and high-affinity nitrate transport system (HATS), respectively. Many genes involved in LATs and HATs have been cloned and studied in multiple plant species, especially in model plant Arabidopsis.
Five genes involved in nitrate transport which belonged to NRT2gene family have been cloned in rice, however, the functional properties study of these genes was unclear. In this thesis, we focus on the high-affinity nitrate transport gene OsNRT2.3a/b in rice. In order to understand the in planta function of OsNRT2.3a/b, the over-expression and RNA interference transgenic mutant plants were obtained. RT-PCR, qRT-PCR, TAIL-PCR, Southern blot and Western blot methods were employed to identify the transgenic plants. The nitrate uptake and translocation of transgenic plants were analyzed by15N labeled NH4+and NO3". The yields of transgenic plants and the potential genes directly or indirectly regulated by OsNRT2.3a/b were also analyzed in the thsis. The main results were as follows:
1. Sequence alignment between genomic and cDNA sequences showed that one gene OsNRT2.3was mRNA spliced into two genes, OsNRT2.3a and OsNRT2.3b. There was only90bp difference between OsNRT2.3a and OsNRT2.3b. The results of rice chip database showed that OsNRT2.3a/b were mainly expressed in roots and little in shoots under field condition. Also, the expression level was increaseed gradually as the plant growth, up to the most at the stage of vegetative growth to reproductive growth. The protoplast subcellular localization showed that OsNRT2.3a and OsNRT2.3b were both localized on plasma membrane.
2. Over-expression of OsNRT2.3a/b and knock-down of OsNRT2.3a transgenic plants were successfully obtained through transgenic manipulation. RT-PCR, TAIL-PCR, Southern blot and Western blot methods were employed to identify the stably inherited transgenic lines.
3. There was no significant difference in phenotype when over-expression OsNRT2.3a alone; but the OsNRT2.3b over-expression plants were obviously higher and bigger than WT. Expression patterns of OsNRT2s and OsNAR2.1were analyzed in over-expression transgenic plants. OsNRT2.3a over expression did not affect the expression of OsNRT2.1, OsNRT2.2and OsNAR2.1. The expression of OsNRT2.1, OsNRT2.3a and OsNAR2.1were up-regulated in OsNRT2.3b over expression plants, however, the expression of OsNRT2.4was not affected. Over expression of OsNRT2.3a alone could not improve the growth of rice plants and enhance the nitrate uptake in rice. The biomass of OsNRT2.3b over expression plants was increased a lot compared with WT, and the whole plant nitrogen content of OsNRT2.3b over expression plants was significantly higher than WT, but the total nitrogen concentration was lower than WT under low nitrogen condition.
4. The field experiment with different nitrogen level showed that OsNRT2.3b over expression plants grow better than WT in all nitrogen level, the yields and nitrogen use efficiency were higher than WT both. However, delaying Heading stage of OsNRT2.3b over expression plants resulted in longer growth period than WT. Further study of the growth period difference between OsNRT2.3b over expression plants and WT showed that over expression of OsNRT2.3b in rice not only increased accumulation and transfer amounts of dry matter but also increased the ratio of dry matter transfer to grain and contribution ratio of transferred dry matter to grain. It also revealed that over expression of OsNRT2.3b in rice not only increased accumulation and transfer amounts of nitrogen but also increased the ratio of nitrogen transfer to grain and contribution ratio of transferred nitrogen to grain.
5. The analyses of OsNRT2.3a RNAi mutants showed that knockdown of OsNRT2.3a did not significantly affect nitrate uptake by rice roots with low nitrate supply. However, knockdown of OsNRT2.3a in rice suppressed nitrate transport from root to shoot under low nitrate condition, resulted in nitrate accumulation in rice roots and down regulation of OsNRT2.1/2.2, which decreased nitrate uptake by rice roots.
6. The results of the whole-genome expression of OsNRT2.3a over expression plants and OsNRT2.3a knockdown mutants showed that Carbon and Nitrogen cycle was suppressed in osnrt2.3a knockdown mutants due to nitrogen deficiency in shoots, furthermore, knockdown of OsNRT2.3a affected the plants photosynthesis and resulted in decreasing accumulation of dry matter and growth retardation. The results of the whole-genome expression of OsNRT2.3b over expression plants showed that suppressing photorespiration appropriately and improving antioxidant activity in OsNRT2.3b over expression plants may resulted in the delayed aging and increased biomass and yields.
Taken together, high-affinity nitrate transporter gene OsNRT2.3a was a key factor in nitrate transport from root to shoot. Over expression of OsNRT2.3b can improve nitrate transportation significantly, and then improve nitrogen use efficiency and yields in rice.
引文
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