Shi Jinrui, Wang Hongyu, Wu Yunsheng, Hazebroek Jan, Meeley Robert B, Ertl David S
Pioneer Hi-Bred International, P.O. Box 1004, Johnston, Iowa 50131, USA.
Plant Physiol. 2003 Feb;131(2):507-15. doi: 10.1104/pp.014258.
Reduced phytic acid content in seeds is a desired goal for genetic improvement in several crops. Low-phytic acid mutants have been used in genetic breeding, but it is not known what genes are responsible for the low-phytic acid phenotype. Using a reverse genetics approach, we found that the maize (Zea mays) low-phytic acid lpa2 mutant is caused by mutation in an inositol phosphate kinase gene. The maize inositol phosphate kinase (ZmIpk) gene was identified through sequence comparison with human and Arabidopsis Ins(1,3,4)P(3) 5/6-kinase genes. The purified recombinant ZmIpk protein has kinase activity on several inositol polyphosphates, including Ins(1,3,4)P(3), Ins(3,5,6)P(3), Ins(3,4,5,6)P(4), and Ins(1,2,5,6)P(4). The ZmIpk mRNA is expressed in the embryo, the organ where phytic acid accumulates in maize seeds. The ZmIpk Mutator insertion mutants were identified from a Mutator F(2) family. In the ZmIpk Mu insertion mutants, seed phytic acid content is reduced approximately 30%, and inorganic phosphate is increased about 3-fold. The mutants also accumulate myo-inositol and inositol phosphates as in the lpa2 mutant. Allelic tests showed that the ZmIpk Mu insertion mutants are allelic to the lpa2. Southern-blot analysis, cloning, and sequencing of the ZmIpk gene from lpa2 revealed that the lpa2-1 allele is caused by the genomic sequence rearrangement in the ZmIpk locus and the lpa2-2 allele has a nucleotide mutation that generated a stop codon in the N-terminal region of the ZmIpk open reading frame. These results provide evidence that ZmIpk is one of the kinases responsible for phytic acid biosynthesis in developing maize seeds.
降低种子中的植酸含量是几种作物遗传改良的一个理想目标。低植酸突变体已被用于遗传育种,但尚不清楚哪些基因导致了低植酸表型。采用反向遗传学方法,我们发现玉米(Zea mays)低植酸lpa2突变体是由肌醇磷酸激酶基因突变引起的。通过与人及拟南芥Ins(1,3,4)P(3) 5/6-激酶基因进行序列比较,鉴定出了玉米肌醇磷酸激酶(ZmIpk)基因。纯化的重组ZmIpk蛋白对几种肌醇多磷酸具有激酶活性,包括Ins(1,3,4)P(3)、Ins(3,5,6)P(3)、Ins(3,4,5,6)P(4)和Ins(1,2,5,6)P(4)。ZmIpk mRNA在胚中表达,胚是玉米种子中植酸积累的器官。从一个Mutator F(2)家系中鉴定出了ZmIpk Mutator插入突变体。在ZmIpk Mu插入突变体中,种子植酸含量降低了约30%,无机磷酸盐增加了约3倍。这些突变体也像lpa2突变体一样积累了肌醇和肌醇磷酸。等位性测试表明,ZmIpk Mu插入突变体与lpa2是等位的。对lpa2的ZmIpk基因进行Southern杂交分析、克隆和测序表明,lpa2-1等位基因是由ZmIpk基因座中的基因组序列重排引起的,而lpa2-2等位基因有一个核苷酸突变,该突变在ZmIpk开放阅读框的N端区域产生了一个终止密码子。这些结果提供了证据,证明ZmIpk是发育中的玉米种子中负责植酸生物合成的激酶之一。