Identification And Cloning Of DNA Segments That Lie The Boundaries Of Known Maize DNA Sequences In T9Generation Rice Variants By Perating Maize DNA Into Rice

Large numbers of rice variant materials were obtained in our laboratory, which werederived from transferring maize genomic DNA fragments into the receptor of rice by distantmolecule hybridization., We have obtained many stable heritable rice variant lines which havesimilar characteristics to donor maize via several times of self-crossbreed. Rice variant line131-5-2which has stably inherited to the ninth generation was researched by identificationand cloning of maize DNA fragments from it in order to provide the theoretical data fordistant molecule hybridization technology that creates new rice germplasm by the transfer oflarge maize DNA fragment into the rice genome.A maize DNA sequence fragment named P5M3-131-349of349bp was cloned fromT7generation of rice variation line131-5-2by AFLP marker technique in our laboratory. Inthis study, the fragment sequence P5M3-131-349was further detected in the T9generation ofrice variation line131-5-2by Nested PCR.The target-DNA fragment of approximately212bpwas amplified from positive control maize and T9generation of rice variant line131-5-2, butnot from negative control rice. The sequence alignment results show that the amino acidshomology of the target-DNA fragment is99.5%between T9generation of rice variant line131-5-2and the positive control maize, and100%between T9and T7generation. The resultsindicate that the target sequence of T7generation derived from maize exists in the T9generation of rice variation line131-5-2and not only has it been fully integrated into thegenome of rice variation line131-5-2, but also it is stably inherited in rice variationline131-5-2.The sequence alignment results show that P5M3-131-349sequence is located on the10th chromosome in maize via MaizeGDB Blast. The primer pairs of HF/HR were designedbased on the10th chromosome sequencing results published from the MaizeGDB databaseand used to amplify3'flanking sequence of P5M3-131-349. The target fragment of541bpwas amplified from positive control maize and T9generation of rice variation line131-5-2,but not from negative control rice. The sequence of P5M3-349-541which was amplified fromT9generation of rice variation line131-5-2has100%amino acid homology compared withwhich of the positive control maize. P5M3-349-541is a597bp sequence combined thesequences of P5M3-349-541and P5M3-131-349by the DNAStar software between. Quering the sequence P5M3-349-597to the database of Nucleotide collection (Nr/nt) on the web ofNCBI show that it has100%similarity with the maize mRNA (EU963081.1) which translatesa protein called Zea mays cysteine-type endopeptidase/ubiquitin thiolesterase. The sequenceP5M3-349-597locates between nucleotides260and856in the EU963081.1mRNA.Protein structure prediction result shows that tcysteine-type endopeptidase/ubiquitinthioesterase protein containing495aa has two hydrophobic transmembrane regions,whichmay locates in the membrane of the plasma membrane, chloroplast membrane, Golgiapparatus or endoplasmic network. It remains to be studied whether the protein is related tothe characteristics of aerial roots, purple stems, purple leaf margin, purple sheath in ricevariation line131-5-2.