Towards the biofortification of banana fruit for enhanced micronutrient content

In Uganda, vitamin A deficiency (VAD) and iron deficiency anaemia (IDA) are major public health problems with between 15-32% of children under 5 years of age showing VAD and 73% being anaemic. This is largely due to the fact that the staple food crop of the country, banana, is low in pro-vitamin A and iron, therefore leading to dietary deficiencies. Although worldwide progress has been made to control VAD and IDA through supplementation, food fortification and diet diversification, their long term sustainability and impact in developing countries such as Uganda is limited. The approach taken by researchers at Queensland University of Technology (QUT), Australia, in collaboration with the National Agricultural Research Organization (NARO), Uganda, to address this problem, is to generate consumer acceptable banana varieties with significantly increased levels of pro-vitamin A and iron in the fruit using genetic engineering techniques. Such an approach requires the use of suitable, well characterised genes and promoters for targeted transgene expression. Recently, a new banana phytoene synthase gene (APsy2a) involved in the synthesis of pro-vitamin A (pVA) carotenoids was isolated from a high â-carotene banana (F’ei cv Asupina). In addition, sequences of banana ferritin, an iron storage protein, have been isolated from Cavendish banana. The aim of the research described in this thesis was to evaluate the function of these genes to assess their suitability for the biofortification of banana fruit. In addition, a range of banana-derived promoters were characterised to determine their suitability for controlling the expression of transgenes in banana fruit. Due to the time constraints involved with generating transgenic banana fruit, rice was used as the model crop to investigate the functionality of the banana-derived APsy2a and ferritin genes. Using Agrobacterium-mediated transformation, rice callus was transformed with APsy2a +/- the bacterial-derived carotene desaturase gene (CrtI) each under the control of the constitutive maize poly-ubiquitin promoter (ZmUbi) or seed-specific rice glutelin1 (Gt1) promoter. The maize phytoene synthase (ZmPsy1) gene was included as a control. On selective media, with the exception of ZmUbi-CrtI-transgenic callus, all antibiotic resistant callus displayed a yellow-orange colour from which the presence of â-carotene was demonstrated using Raman spectroscopy. Although the regeneration of plants from yellow-orange callus was difficult, 16 transgenic plants were obtained and characterised from callus transformed with ZmUbi-APys2a alone. At least 50% of the T1 seeds developed a yellow-orange coloured callus which was found to contain levels of â-carotene ranging from 4.6-fold to 72-fold higher than that in non-transgenic rice callus. Using the seed-specific Gt1 promoter, 38 transgenic rice plants were generated from APsy2a-CrtI-transformed callus while 32 plants were regenerated from ZmPsy1-CrtI-transformed callus. However, when analysed for presence of transgene by PCR, all transgenic plants contained the APsy2a, ZmPsy1 or CrtI transgene, with none of the plants found to be co-transformed. Using Raman spectroscopy, no â-carotene was detected in-situ in representative T1 seeds. To investigate the potential of the banana-derived ferritin gene (BanFer1) to enhance iron content, rice callus was transformed with constitutively expressed BanFer1 using the soybean ferritin gene (SoyFer) as a control. A total of 12 and 11 callus lines independently transformed with BanFer1 and SoyFer, respectively, were multiplied and transgene expression was verified by RT-PCR. Pearl’s Prussian blue staining for in-situ detection of ferric iron showed a stronger blue colour in rice callus transformed with BanFer1 compared to SoyFer. Using flame atomic absorption spectrometry, the highest mean amount of iron quantified in callus transformed with BanFer1 was 30-fold while that obtained using the SoyFer was 14-fold higher than the controls. In addition, ~78% of BanFer1-transgenic callus lines and ~27% of SoyFer-transgenic callus lines had significantly higher iron content than the non-transformed controls. Since the genes used for enhancing micronutrient content need to be expressed in banana fruit, the activity of a range of banana-derived, potentially fruit-active promoters in banana was investigated. Using uidA (GUS) as a reporter gene, the function of the Expansin1 (MaExp1), Expansin1 containing the rice actin intron (MaExp1a), Expansin4 (MaExp4), Extensin (MaExt), ACS (MaACS), ACO (MaACO), Metallothionein (MaMT2a) and phytoene synthase (APsy2a) promoters were transiently analysed in intact banana fruit using two transformation methods, particle bombardment and Agrobacterium-mediated infiltration (agro-infiltration). Although a considerable amount of variation in promoter activity was observed both within and between experiments, similar trends were obtained using both transformation methods. The MaExp1 and MaExp1a directed high levels of GUS expression in banana fruit which were comparable to those observed from the ZmUbi and Banana bunchy top virus-derived BT4 promoters that were included as positive controls. Lower levels of promoter activity were obtained in both methods using the MaACO and MaExt promoters while the MaExp4, MaACS, and APsy2a promoters directed the lowest GUS activity in banana fruit. An attempt was subsequently made to use agro-infiltration to assess the expression of pVA biosynthesis genes in banana fruit by infiltrating fruit with constructs in which the ZmUbi promoter controlled the expression of APsy2a +/- CrtI, and with the maize phytoene synthase gene (ZmPsy1) included as a control. Unfortunately, the large amount of variation and inconsistency observed within and between experiments precluded any meaningful conclusions to be drawn. The final component of this research was to assess the level of promoter activity and specificity in non-target tissue. These analyses were done on leaves obtained from glasshouse-grown banana plants stably transformed with MaExp1, MaACO, APsy2a, BT4 and ZmUbi promoters driving the expression of the GUS gene in addition to leaves from a selection of the same transgenic plants which were growing in a field trial in North Queensland. The results from both histochemical and fluorometric GUS assays showed that the MaExp1 and MaACO promoters directed very low GUS activities in leaves of stably transformed banana plants compared to the constitutive ZmUbi and BT4 promoters. In summary, the results from this research provide evidence that the banana phytoene synthase gene (APsy2a) and the banana ferritin gene (BanFer1) are functional, since the constitutive over-expression of each of these transgenes led to increased levels of pVA carotenoids (for APsy2a) and iron content (for BanFer1) in transgenic rice callus. Further work is now required to determine the functionality of these genes in stably-transformed banana fruit. This research also demonstrated that the MaExp1 and MaACO promoters are fruit-active but have low activity in non-target tissue (leaves), characteristics that make them potentially useful for the biofortification of banana fruit. Ultimately, however, analysis of fruit from field-grown transgenic plants will be required to fully evaluate the suitability of pVA biosynthesis genes and the fruit-active promoters for fruit biofortification.

高赖氨酸蛋白基因的克隆及其转基因水稻植株的获得

水稻、玉米、小麦和大麦等许多主要禾本科作物的第一限制性氨基酸是赖氨酸。本文将一个来源于四棱豆的高赖氨酸蛋白基因导入水稻，以研究通过转基因改善蛋白质的可能，获得有经济价值和社会意义的转基因作物。 构建了含有高赖氨酸蛋白基因（Lys）、gus基因及植物选择标记潮霉素磷酸转移酶基因（hpt）的植物表达载体pBRLys;在pBRLys中，该高赖氨酸蛋白基因由目前已知最强的单子叶植物启动子玉米Ubiquitin 1启动子调控。用基因枪轰击法将pBRLys导入水稻幼胚或幼胚诱导的愈伤组织。共得到36株潮霉素抗性再生植株，经分子检测有22株为转基因植株。 实验中对影响水稻转化、再生和移栽一些条件进行了研究。从潮霉素筛选浓度、愈伤组织干燥处理、光照对分化的影响、多效唑的影响和移栽环境等做了一些简化和改善。 PCR检测、PCR-Southern杂交和Southern杂交表明潮霉素基因和Lys基因已经整合到转基因水稻的基因组中，外源基因在转基因水稻基因组中以1个拷贝以上的形式存在。同时，GUS组织化学染色表明转基因水稻植株的叶、茎和根中都有gus基因的表达。 初步对5株转基因植株进行赖氨酸含量测定，结果表明：与非转化对照相比，有两棵植株赖氨酸含量提高，分别增加6.0%和12.4%。对更多抗性转化植株的分子检测、GUS分析和赖氨酸含量测定正在进行之中。

MADS-box基因在单子叶植物花发育中的功能研究

第一部分 水稻E类MADS-box 基因在花发育中的功能分析 MADS-box 基因是一个大的转录因子家族，在花发育过程中起重要作用。根据对双子叶模式植物拟南芥、金鱼草和矮牵牛遗传突变体的研究，提出了花发育的ABCDE模型。该模型认为：A、B、C、D、E代表了5类功能不同的花器官特征基因，单独或联合控制花器官的发育。A类基因控制萼片的发育;A、B和E类基因控制花瓣的发育;B、C和E类基因控制雄蕊的发育;C和E类基因控制心皮的发育;D类基因控制胚珠的发育;A和C类基因相互抑制。在这5类基因中，E类基因的功能较为复杂，它不仅是花器官特征基因，而且具有花分生组织决定性（Floral meristem determinency）。在单子叶植物中，E类基因的功能发生了很大的分化。水稻是单子叶植物的模式植物，水稻中至少有5个E类基因，分别是OsMADS1、OsMADS5、OsMADS7、OsMADS8和OsMADS34，在这5个E类基因中，除了对OsMADS1基因有较深入的研究外，对其它几个E类基因的功能了解甚少。我们在现有的研究基础上，根据对双子叶植物中E类基因的研究结果，以OsMADS8基因为出发点，利用组织原位杂交，RNAi技术对水稻中的E类基因进行了深入的研究。结果表明：OsMADS8/7基因早在花序枝梗分生组织原基就有转录，随着小穗的生长发育，逐渐集中在小穗分生组织原基，小花分生组织原基，浆片、雄蕊和心皮中表达;在胚珠形成时，内外珠被有很强的杂交信号，而且在幼胚和胚乳中也有表达。OsMADS5在幼花时期，四轮花器官均有表达，在小穗发育后期及受精后的表达方式与OsMADS8/7基因相同。OsMADS8基因被抑制后，转基因植株没有任何表型变化，说明很可能有其它E类基因弥补了OsMADS8基因的功能缺失;当同时抑制其它E类基因的表达时，转基因植株抽穗期明显延长，四轮花器官的发育均受到影响：稃片类似叶片状;浆片转变为稃片类的结构;雄蕊没有花粉;心皮具有了稃片的特点;没有胚珠结构的形成，同时失去了花分生组织决定性，在心皮的部位产生了新的花器官或花分生组织逆转为花序分生组织。说明水稻四轮花器官及胚珠的正常发育需要E类基因的参与，但其功能与双子叶植物如拟南芥，西红柿、矮牵牛等直系同源基因相比已经发生变化;水稻中的E类基因在维持花分生组织特征性方面起重要作用;另外对抽穗期有影响。 第二部分 玉米MADS-box基因ZAG2转录调控区的研究 基因的时空表达受基因中的顺式作用元件及其反式作用因子调控。顺式作用元件由位于基因编码区上游的启动子区域和位置不确定的增强子区域组成。顺式作用元件对基因表达的开启至关重要。MADS-box 基因编码一类控制花器官发育的转录因子，在花的发育过程中顺序表达。MADS-box 基因突变，花器官发生同源异型转换。研究MADS-box 基因的调控序列可以进一步揭示影响基因时空表达的内外因素。ZAG2是玉米MADS-box 基因中的D类基因，控制胚珠的发育，在胚珠和心皮的内表面特异表达。ZAG2基因有7个外显子和6个内含子。我们从玉米基因组分离到了ZAG2基因翻译起始点上游3040bp的序列，并利用5’-RACE方法鉴定出了转录起始点的位置。序列比较发现，在 5’-UTR内有一个1299bp的内含子，这个内含子可能对基因的表达有调控作用，因此构建了两个与GUS基因融合的表达载体：一个是pZAG2-1::GUS，包括翻译起始点以上所有的调控序列;另一个是pZAG2-2::GUS，去掉了5’-UTR中的内含子序列，转化水稻。结果这两个构建都没有使GUS基因在正确的位置表达。pZAG2-1::GUS构建在心皮基部类似花托的部位及稃片顶端着色，pZAG2-2::GUS构建在内外稃片沿稃脉的部位有很强的着色，说明翻译起始点上游的调控序列不足以使基因正常表达。两个构建着色方式不同，可能pZAG2-1::GUS构建在5’-UTR部分含有抑制ZAG2基因在稃片表达的顺式元件，或者启用了在5’-UTR中的转录起始点，因为在5’-UTR的内含子中也有一个很典型的TATA-box。我们推测，在ZAG2基因编码区的第一内含子可能存在另外一些使基因正常表达的增强元件，需要进一步的序列缺失实验加以验证。

转Rubisco 活化酶大型同工酶基因(rca)水稻的光合作用特性研究

Rubisco 是催化光合暗反应第一步反应的酶，是唯一能将CO2 转变成碳水化合物的酶，由它固定和最后转化成的碳水化合物提供了植物、动物和微生物的食物和能量。但是，Rubisco 催化该反应的效率十分低，使之成为光合作用的限速步骤。由于Rubisco 的合成和催化过程十分复杂，人们很难通过直接改造Rubisco 提高植物固定CO2 的能力。而Rubisco 活化酶能活化Rubisco，使植物在生理CO2 浓度下具有最大的CO2 同化速率，因此研究活化酶有重要意义。水稻活化酶有2 个同工酶，大型同工酶比小型同工酶C 端多37 个氨基酸，其中包括两个Cys 残基。这两个Cys 残基的存在使活化酶大型同工酶对ADP 的存在更加敏感，其活性在硫氧还蛋白的介导下能被基质中氧化还原状态的变化所调节。由于活化酶大型同工酶对调节Rubisco 的活性具有的这种特殊作用，在本研究中，将活化酶大型同工酶rca基因用正义和反义引入水稻基因组，获得了过量表达活化酶大型同工酶基因和反义抑制活化酶基因表达的转基因植株，对其光合作用进行了生理和生化分析。 本研究的主要结果如下： Rubisco 活化酶大型同工酶基因的克隆：从水稻镇恢249 中克隆了1525 bp 的活化酶大型同工酶cDNA 序列。经过测序，它与报道的粳稻品种活化酶大型同工酶cDNA 序列（rca）完全相同。 构建了4 个植物表达载体：3 个为过量表达rca的载体，分别是pCBUbirca，pCBSrca 和 pCBSUbirca ，其中rca分别在水稻中高效表达的玉米Ubiquitin 启动子、受光调控的Rubisco 小亚基基因启动子和由这两个启动子构成的双启动子控制下表达; 1 个在Ubiquitin 启动子控制的反义rca载体，即 pCBUbi-antirca。 获得了转化rca的水稻再生植株：用日本晴，台北309，武育梗7 号和籼稻品种培矮64S 水稻成熟种子诱导愈伤组织。用改良的农杆菌浸染法将rca基因转化这些愈伤组织，在潮霉素筛选压力下获得抗性愈伤组织，经过2 天的干燥处理后，转入到含山梨醇的高渗分化培养基上培养，能迅速获得大量的芽和转化体再生植株。 获得了转rca基因的水稻植株：抗性愈伤组织和再生水稻幼苗的叶片经GUS 染色呈蓝黑色。PCR 扩增转基因水稻基因组内的潮霉素基因和rca，大部分转基因水稻中含有841 bp 的潮霉素基因片段和1525 bp 长的rca cDNA 片段。251 粒T1 代转基因水稻种子中189粒呈现潮霉素抗性，抗性种子/非抗性种子的比率约为3：1，接近孟德尔分离规律。Southern杂交表明rca序列已整合到水稻基因组，一般含1－2个拷贝。Western 杂交显示Rubisco 活化酶含量在转pCBUbi －antirca 的水稻中和对照比，几乎看不出，被反义抑制;转pCBUbirca 的水稻与对照含量相差无几;转pCBSUbirca，pCBSrca 载体的水稻中活化酶的含量比对照有极显著的增加。 T1 代转rca水稻的光合作用发生显著变化：转pCBSrca 和pCBSUbirca 的水稻在饱和光强下的Rubisco 初始活性、羧化效率、光合速率都明显高于对照，但是表观量子效率、色素含量和Rubisco 总活性与对照相似。两者相比，前者比后者更高;转反义rca（pCBUbi-antirca）基因的水稻饱和光强下的光合速率、表观量子效率、羧化效率、Rubisco 初始活性明显降低，色素含量和Rubisco 总活性基本不变;转pCBUbirca 的水稻中，光合作用的各项参数与对照基本相似。 T1 代转rca水稻的叶绿素荧光明显改变：转pCBSrca 和pCBSUbirca 的水稻ΦPSII 的值明显高于对照，而且前者qP 的值明显高于对照。两者相比，前者的ΦPSII 和qP 的值比后者高;转反义rca的水稻ΦPSII，F′v/F′m，qP 值和对照比都明显降低，但qN 的值升高;转pCBUbirca 载体的水稻中，叶绿素荧光的各项参数与对照基本相似。 转rca基因的水稻生长发育的变化：转pCBUbirca 载体的水稻整个生长发育过程与对照相似;转化pCBSrca 和pCBSUbirca 载体的水稻和对照比，植株高大，生长发育速度加快，抽穗、开花和结籽的时间提前。两者本身相比，前者比后者明显;转反义rca（pCBUbi-antirca）基因的水稻生长发育延迟，植株矮小，种子败育。 由上可见，Rubisco 活化酶大型同工酶rca基因在Rubisco 小亚基基因启动子、Ubiquitin 基因启动子和Rubisco 小亚基基因启动子共同控制下正义转入水稻的转基因植物光合作用的参数最好，光合效率提高，植物表型最好，生长发育加快，提前开花结籽。这一研究可能为获得高光合效率和高产量的水稻奠定了基础。

谷子和甘蔗C4型磷酸烯醇式丙酮酸羧化酶基因的克隆及其转基因水稻光合效率的提高

水稻是我国重要的粮食作物之一，它是一种典型的C3植物。与其它C3作物不一样的是，水稻的生长需要相对较高的温度和充足的阳光照射。然而高温和高光强的生长环境更加适合于C4植物的生长，更加有利于发挥C4植物高光合效率的特点。因此本论文希望将C4植物中固定CO2的酶磷酸烯醇式丙酮酸羧化酶基因导入水稻，获得一种更加适合高温和高光强生活环境的“C4型”水稻，这对于提高水稻的产量，满足人口增长对粮食需求具有重大意义。 本论文从C4植物谷子和甘蔗中克隆了其C4型磷酸烯醇式丙酮酸羧化酶cDNA基因，获得了具有自主知识产权的基因克隆，并将它们导入粳稻品种中花8号，进而对转基因材料的光合生理特性进行了研究。结果如下： 首次从谷子中得到了ppc基因两个cDNA克隆，分别命名为Mppc1和Mppc2。前者是一个C3型的ppc基因，它可能属于在根中特异表达的C3-2型ppc基因;后者是在绿色叶片中大量表达的C4型ppc基因。它们所编码的蛋白的氨基酸残基数分别为961和964，序列同源性为82.5%。C4型PEPC多出的3个氨基酸位于N末端。利用RACE的方法我们得到了谷子C4型ppc基因完整的cDNA序列，包括63bp的5'非编码区，2895bp的编码区和256bp的3'非编码区。 首次获得了甘蔗C4型ppc基因完整的cDNA序列的克隆，命名为Sppc。它包括95bp的5'非编码区、2886bp的编码区，和224bp的3'非编码区。 利用所克隆的基因，分别连上强组成型启动子Ubiquitin启动子和强光调控启动子Rubisco小亚基启动子后，再插入两个标记基因不同的表达载体pCB和pPCB的多克隆位点中，构建了八个含有外源ppc基因的植物表达载体pCB-Pubi-Mppc、pCB-Pubi-Sppc、pCB-PrbcS-Mppc、pCB-PrbcS-Sppc、pPCB-Pubi-Mppc、pPCB-Pubi-Sppc、pPCB-PrbcS-Mppc和pPCB-PrbcS-Sppc。再加上含有玉米完整的C4型ppc 核基因的载体pCB-ZMppc，共有9个载体。利用农杆菌介导法进行了水稻的转化，各个载体都获得了大量的转基因植株。对标记基因潮霉素磷酸转移酶基因hpt和磷酸甘露糖异构酶基因pmi以及导入的目的ppc基因的PCR扩增检测，结果显示绝大多数转基因植株都能扩增出目的片段，而未转化的植株则没有扩增产物。对部分转基因水稻的Southern和Western杂交以及RT-PCR分析都表明，无论从DNA水平、mRNA水平，还是从蛋白质水平上都证明外源ppc基因都成功地导入了水稻，并获得了正确的表达。 对各载体转基因植株PEPC活性大规模的测定表明，转入玉米完整C4型PEPC核基因（有内含子）的水稻表现出极大的表达效率，大多数转基因材料的PEPC活性为对照的10-20倍，其活性最高可达到对照的44倍。转入谷子和甘蔗PEPC基因cDNA的水稻，表达的效率很低，多数材料活性增加仅为对照的2-5倍，但也有极少数材料活性增加了10倍以上。用Rubisco小亚基启动子控制的ppc基因在水稻的表达活性要略高于Ubiquitin启动子控制的ppc基因。以上结果说明ppc基因的内含子在其转录或mRNA的稳定上起着重要作用。 对部分转基因材料气体交换特征的研究发现，随着转基因水稻PEPC活性的增加，净光合速率也有逐渐增加的趋势。其中PEPC活性最大的ZM24株系的三个单株净光合速率比对照增加了39.8%、13.7%和28.6%，而它们的PEPC活性比对照分别增加了21.2、21.9和23.6倍。 转PEPC水稻的净光合速率与气孔导度具有显著的相关性。这说明表达的外源ppc 基因产物PEPC参与了转基因水稻的气孔运动，使气孔开放程度增加。更有意义的是过表达PEPC的水稻具有更高的水分利用效率，这就增加了其耐旱能力。在光抑制条件下转基因水稻也具有更高的光合能力。这些特征表明转ppc基因的水稻比对照更加适合于水稻高温高光强和干旱的原生环境。

Oryza sativa PSK gene encodes a precursor of phytosulfokine-α, a sulfated peptide growth factor found in plants

Phytosulfokine-α [PSK-α, Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln], a sulfated mitogenic peptide found in plants, strongly promotes proliferation of plant cells in culture at very low concentrations. Oryza sativa PSK (OsPSK) cDNA encoding a PSK-α precursor has been isolated. The cDNA is 725 base pairs long, and the 89-aa product, preprophytosulfokine, has a 22-aa hydrophobic region that resembles a cleavable leader peptide at its NH2 terminus. The PSK-α sequence occurs only once within the precursor, close to the COOH terminus. [Ser4]PSK-α was secreted by transgenic rice Oc cells harboring a mutated OsPSK cDNA, suggesting proteolytic processing from the larger precursor, a feature commonly found in animal systems. Whereas PSK-α in conditioned medium with sense transgenic Oc cells was 1.6 times as concentrated as in the control case, antisense transgenic Oc cells produced less than 60% of the control level. Preprophytosulfokine mRNA was detected at an elevated constitutive level in rice Oc culture cells on RNA blot analysis. Although PSK-α molecules have never been identified in any intact plant, reverse transcription–PCR analysis demonstrated that OsPSK is expressed in rice seedlings, indicating that PSK-α may be important for plant cell proliferation both in vitro and in vivo. DNA blot analysis demonstrated that OsPSK homologs may occur in dicot as well as monocot plants.

Overexpression and characterization of rice Phosphorus-Starvation Tolerance 1 gene and its sorghum and maize homologs in transgenic tobacco.

2016

High-efficiency silencing of a β-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation

Two transgenic callus lines of rice, stably expressing a β-glucuronidase (GUS) gene, were supertransformed with a set of constructs designed to silence the resident GUS gene. An inverted-repeat (i/r) GUS construct, designed to produce mRNA with self-complementarity, was much more effective than simple sense and antisense constructs at inducing silencing. Supertransforming rice calluses with a direct-repeat (d/r) construct, although not as effective as those with the i/r construct, was also substantially more effective in silencing the resident GUS gene than the simple sense and antisense constructs. DNA hybridisation analyses revealed that every callus line supertransformed with either simple sense or antisense constructs, and subsequently showing GUS silencing, had the silence-inducing transgenes integrated into the plant genome in inverted-repeat configurations. The silenced lines containing i/r and d/r constructs did not necessarily have inverted-repeat T-DNA insertions. There was significant methylation of the GUS sequences in most of the silenced lines but not in the unsilenced lines. However, demethylation treatment of silenced lines with 5-azacytidine did not reverse the post-transcriptional gene silencing (PTGS) of GUS. Whereas the levels of RNA specific to the resident GUS gene were uniformly low in the silenced lines, RNA specific to the inducer transgenes accumulated to a substantial level, and the majority of the i/r RNA was unpolyadenylated. Altogether, these results suggest that both sense- and antisense-mediated gene suppression share a similar molecular basis, that unpolyadenylated RNA plays an important role in PTGS, and that methylation is not essential for PTGS.

Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

We report the first successful Agrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph (encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4-fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 carrying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher β-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity comparable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbil-gus plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs. Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T 1 generation.

Transfer of saline tolerance from one strain of rice to another by injection of DNA

Direct injection of genomic DNA from salt tolerant cv. Pokkali into developing floral tillers on IR20 produced transgenic seeds similar to Pokkali in husk colour and which germinated well in 0.2 M NaCl and had a 4-6-fold higher proline content.

Functions for rice RFL in vegetative axillary meristem specification and outgrowth

Roles for the transcription factor RFL in rice axillary meristem development were studied. Its regulatory effects on LAX1, CUC1, and OsPIN3 reveal its functions in axillary meristem specification and outgrowth.Axillary meristems (AMs) are secondary shoot meristems whose outgrowth determines plant architecture. In rice, AMs form tillers, and tillering mutants reveal an interplay between transcription factors and the phytohormones auxin and strigolactone as some factors that underpin this developmental process. Previous studies showed that knockdown of the transcription factor gene RFL reduced tillering and caused a very large decrease in panicle branching. Here, the relationship between RFL, AM initiation, and outgrowth was examined. We show that RFL promotes AM specification through its effects on LAX1 and CUC genes, as their expression was modulated on RFL knockdown, on induction of RFL:GR fusion protein, and by a repressive RFL-EAR fusion protein. Further, we report reduced expression of auxin transporter genes OsPIN1 and OsPIN3 in the culm of RFL knockdown transgenic plants. Additionally, subtle change in the spatial pattern of IR4 DR5:GFP auxin reporter was observed, which hints at compromised auxin transport on RFL knockdown. The relationship between RFL, strigolactone signalling, and bud outgrowth was studied by transcript analyses and by the tillering phenotype of transgenic plants knocked down for both RFL and D3. These data suggest indirect RFL-strigolactone links that may affect tillering. Further, we show expression modulation of the auxin transporter gene OsPIN3 upon RFL:GR protein induction and by the repressive RFL-EAR protein. These modified forms of RFL had only indirect effects on OsPIN1. Together, we have found that RFL regulates the LAX1 and CUC genes during AM specification, and positively influences the outgrowth of AMs though its effects on auxin transport.

Global transcript analysis of rice leaf and seed using SAGE technology.

We have compiled two comprehensive gene expression profiles from mature leaf and immature seed tissue of rice (Oryza sativa ssp. japonica cultivar Nipponbare) using Serial Analysis of Gene Expression (SAGE) technology. Analysis revealed a total of 50 519 SAGE tags, corresponding to 15 131 unique transcripts. Of these, the large majority (approximately 70%) occur only once in both libraries. Unexpectedly, the most abundant transcript (approximately 3% of the total) in the leaf library was derived from a type 3 metallothionein gene. The overall frequency profiles of the abundant tag species from both tissues differ greatly and reveal seed tissue as exhibiting a non-typical pattern of gene expression characterized by an over abundance of a small number of transcripts coding for storage proteins. A high proportion ( approximately 80%) of the abundant tags (> or = 9) matched entries in our reference rice EST database, with many fewer matches for low abundant tags. Singleton transcripts that are common to both tissues were collated to generate a summary of low abundant transcripts that are expressed constitutively in rice tissues. Finally and most surprisingly, a significant number of tags were found to code for antisense transcripts, a finding that suggests a novel mechanism of gene regulation, and may have implications for the use of antisense constructs in transgenic technology.

Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm

Background
Rice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g−1 Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g−1 Fe in endosperm.

Methodology/Principal Findings
Three populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g−1 Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g−1 Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm.

Conclusions
The OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.