Introduction of Wx transgene into rice wx mutants leads to both high- and low-amylose rice

The Waxy (Wx) gene encodes a granule-bound starch synthase (GBSS) that plays a key role in the amylose synthesis of rice and other plant species. Two functional Wx alleles of rice exist: Wx(a), which produces a large amount of amylose, and Wx(b), which produces a smaller amount of amylose because of the mutation at the 5' splice site of intron 1. Wx(b) is largely distributed in Japonica cultivars, and high amylose cultivars do not exist in Japonica cultivars. We introduced the cloned Wx(a) cDNA into null-mutant Japonica rice (wx). The amylose contents of these transgenic plants were 6-11% higher than that of the original cultivar, Labelle, which carries the Wx(a) allele, although the levels of the Wx protein in the transgenic rice were equal to those of cv. Labelle. We also observed a gene-dosage effect of the Wx(a) transgene on Wx protein expression, but a smaller dosage effect was observed in amylose production with over 40% of amylose content in transgenic rice. Moreover, one transgenic line carrying eleven copies of the transgene showed low levels of Wx expression and amylose in the endosperm. This suggested that the integration of excessive copies of the transgene might lead to gene silencing.     

水稻Wx基因表达调控的研究进展

水稻Wx基因编码颗粒结合淀粉合成酶(GBSS),是控制直链淀粉合成的主效基因。文中主要从转录水平和转录后水平介绍水稻Wx基因表达调控的研究进展,同时介绍转基因、遗传背景以及环境温度对Wx基因表达的影响,并提出Wx基因表达调控研究中一些期待解决的问题。     

Du1, encoding a novel Prp1 protein, regulates starch biosynthesis through affecting the splicing of Wxb pre-mRNAs in rice (Oryza sativa L.)

Starch is the major component of cereal grains. In rice, starch properties determine the eating and cooking quality. The dull endosperm of rice grains is a classical morphological and agronomical trait that has long been exploited for breeding and genetics study. To understand the molecular mechanism that regulates the starch biosynthesis in rice grains, we characterized a classic rice mutant dull endosperm1 (du1) and isolated Du1 through a map-based cloning approach. Du1, encoding a member of pre-mRNA processing (Prp1) family, is expressed mainly in panicles. Du1 specifically affects the splicing efficiency of Wx(b) and regulates starch biosynthesis by mediating the expression of starch biosynthesis genes. Analysis of du1wx shows that Du1 acts upstream of Wx(b). These results strongly suggest that Du1 may function as a regulator of the starch biosynthesis by affecting the splicing of Wx(b) and the expression of other genes involved in the rice starch biosynthetic pathways.     

复合调控Wx与SBE3基因对水稻籽粒直链淀粉含量的影响

颗粒淀粉合成酶（GBSS）和淀粉分支酶3（SBE3）是淀粉合成过程中的两个关键酶,这两个酶主要由耽和SBE3两个基因分别控制,它们的表达量直接影响直链淀粉和支链淀粉的含量比例。为了探讨水稻淀粉关键酶基因耽过量与SBE3干涉复合表达对直链淀粉含量的影响,构建了Wx过量表达与SBE3干涉结合的多基因表达载体,并通过农杆菌介导的方法将其导入日本晴水稻中。经过PCR检测分析获得了65株转基因阳性植株,半定量RT—PCR检测表明转基因株系中Wx基因表达量明显增加,而SBE3基因表达量显著减少。转基因株系籽粒透明度明显降低,直链淀粉含量比野生型的平均高45％,但是千粒重变化不大,与野生型相当。遗传分析表明这些转基因株系多数可稳定遗传。     

控制水稻胚乳淀粉合成代谢若干关键酶基因对花后高温的响应表达

以稻米品质温度敏感型的早籼稻品种嘉早935为材料,利用人工气候箱控温试验和实时荧光定量PCR技术,探讨了不同灌浆温度(日均温分别为22和32 ℃)处理下胚乳淀粉分支酶(SBE)、淀粉去分支酶(DBE)和淀粉合酶(SS)的10个同工型基因(sbe1、sbe3、sbe4、pul、isa1、isa2、isa3、Wx、sss1和sss2a)的相对表达量差异及动态变化特征.结果表明: 淀粉合成相关功能基因对水稻灌浆期高温胁迫的响应表达方式存在明显差异,而且因同工型的类型而不同.在高温处理下,sbe1和sbe3的相对表达量显著下降,二者属于SBE类基因中对高温胁迫较敏感的主要同工型;DBE基因中,pul属于高表达的同工型,而且其对高温胁迫响应比isa1、isa2和isa3敏感;在Wx、sss1和sss2a中,sss2a的相对表达量显著低于sss1和Wx, 但sss2a和sss1对高温胁迫响应比Wx敏感,因此二者可能也是高温胁迫对胚乳淀粉结构进行调控的重要位点,尤其在水稻灌浆的中后期发挥重要作用.     

Excision of Ds produces waxy proteins with a range of enzymatic activities.

The waxy (wx) locus of maize encodes an enzyme responsible for the synthesis of amylose in endosperm tissue. The phenotype of the Dissociation (Ds) insertion mutant wx-m1 is characterized by endosperm sectors that contain different levels of amylose. We have cloned the Wx gene from this allele and from two germinal derivatives, S5 and S9, that produce intermediate levels of amylose. The Ds insertion in wx-m1 is in exon sequences, is 409 bp in length and represents an example of a class of Ds elements that are not deletion derivatives of the Activator (Ac) controlling element. The two germinal derivatives, S5 and S9, lack the Ds element but contain an additional 9 and 6 bp, respectively, at the site of Ds insertion. The level of Wx mRNA and Wx protein in S5 and S9 is essentially the same as in normal endosperm tissue but Wx enzymatic activity is reduced. Thus, the lesions in S5 and S9 lead to the addition of amino acids in the Wx protein, resulting in Wx enzymes with altered specific activities. This work supports the notion that the maize transposable elements may serve a function in natural populations to generate genetic diversity, in this case, proteins with new enzymatic properties.     

OsRRM, a Spen-like rice gene expressed specifically in the endosperm

We used the promoter trap technique to identify a rice plant, named 107^#, in which the β-glucuronidase （GUS） reporter gene was expressed specifically in the endosperm. A single copy of the T-DNA was inserted into the plant genome, and a candidate gene OsRRM was identified by the insertion. The OsRRM promoter directed GUS expression specifically in rice endosperm, analogous to the GUS expression pattern observed in 107^#. OsRRMis a single-copy gene in rice and encodes a nuclear protein containing 1 005 amino-acid residues with two RNA recognition motifs and one Spen paralog and ortholog C-terminal domain. Westem blot analysis confirmed that the OsRRM protein was specifically expressed in rice endosperm. Ectopic expression of OsRRM in transgenic plants led to abnormalities, such as short stature, retarded growth and low fructification rates. Our data, in conjunction with the reported function of Spen genes, implicated OsRRM in the regulation of cell development in rice endosperm.     

同源四倍体水稻籼型突变体Wx基因序列分析及特异识别分子标记的建立

同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因, 根据普通水稻Wx基因设计引物, 扩增测序获得了D4063-1Wx基因的全序列, 并与已报道的Wx基因进行比对分析; 同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生碱基缺失, 导致移码突变, 在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致其序列上酶切位点的变化,对常用限制性内切酶位点分析结果表明, 同源四倍体水稻相对于籼稻和粳稻多了2个sphⅠ酶切位点, 相对于粳稻减少了6个AccⅠ, 增加了4个XbaⅠ, 1个XhoⅠ, 1个PstⅠ和1个SalⅠ酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近, 由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外, 根据D4063-1Wx基因的碱基差异, 推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因, 并可能与其米饭较软等品质相关。本研究还根据D4063-1和籼稻、粳稻的序列差异及D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与作者设计的扩增普通籼稻、粳稻Wx基因的引物F5配合使用, 建立了识别D4063-1的显性和共显性两种检测方式的分子标记, 为快速、准确鉴别低直链淀粉含量突变体D4063-1创造了条件。     

Tissue-Dependent Expression of the Rice wx+ Gene Promoter in Transgenic Rice and Petunia

The waxy (wx) locus, which controls the amylose synthesis, isknown to be expressed specifically in the endosperm and pollen.To study the tissue-specific regulation of the wx⁺ gene, weintroduced a fusion gene that consisted of the upstream sequenceof the wx⁺ gene and the gene for rß-glucuronidase(GUS) into cells of rice (Oryza sativa L.) and petunia (Petuniahybrida L.). GUS activity was examined in the regenerated transgenicrice and petunia plants. In transgenic rice, the upstream sequenceof the wx⁺ gene was sufficient to direct the tissue-specificexpression of GUS in the endosperm and pollen, and the controlof expression was quantitative. By contrast, in transgenic petunia,the same fusion gene was expressed in pollen but not in theendosperm. These results suggest that the putative cis-actingelements that direct pollen-specific expression are common toor similar in both monocotyledonous and dicotyledonous plants,whereas ciy-elements responsible for the endosperm-specificexpression of the rice wx⁺ gene do not function in petunia,in which development of the endosperm differs from that in rice. ⁴Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan